SMB 110: INTRODUCTION TO BACTERIOLOGY (Revised)

Purpose: To equip the learner with knowledge in bacteria, its application and the techniques used in a bacteriology
laboratory

Expected Learning Outcomes: By the end of the course the learner should be able to:

i) Identify the major groups of bacteria

ii) Present experiments to identify bacteria.

iii) Describe the physiology of bacteria.

iv) Explain the various culture and staining techniques of bacteria media types and their applications

Course Content

Bacteria: Isolation and culture of environmental samples, nutritional requirements and bacteriological media, bacterial

growth curve, enumeration of bacteria, physiology, ecology and genetics. Characterization and identification of bacteria.

Major bacterial groups, plant and animal diseases caused by bacteria. Recent advances in bacteriology.

Contact hours: 35 hrs.

mode of Delivery

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Lecture, class dissertations, student presentation, demonstration, illustration and written assignments. Practical: course

relevant experiments.

Instructional Materials/Equipments

Course notes, marker boards, white board markers, dusters, computer and LCD projector. Laboratory equipments, materials

and reagents for practicals

Course assessment

CATs – 30 %; End of semester examination – 70 %

Pass Mark: 40 %

Course Reference Materials:

Core Textbooks

i) Morray, C. B. (2013) The Fundamentals of Bacteriology ISBN-10: 1530527317; ISBN-13: 978-1530527311

ii) Cappuccino, J. and Sherman, N. (2007) Microbiology. A laboratory manual 9th edition .ISBN: 0805325786, ISBN-13

9780805325782

Recommended Textbooks

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 i) Holt, J. G. (1993) Bergey's Manual of Determinative Bacteriology, 9th edition ISBN-10: 0683006037; ISBN-13: 978-

0683006032

ii) Caldwell, D. R. (1995) Microbial and Metabolism. ISBN- 13 9780697171924; ISBN-10:0697171922.

Journals

i) Trends in Microbiology. Elsevier. ISSN: 0966-842X.

ii) Current Microbiology. Springer. ISSN: 0343-8651 (print), ISSN: 1432-0991 (electronic).

iii) Current Opinion in Biotechnology. Elsevier. ISSN: 0958-1669

iv) African Journal of Microbiology Research. Kluwer Academic Publishers. ISSN: 1996-0808.

E-Materials

i) http://www.textbookofbacteriology.net/

ii) https://www.youtube.com/watch?v=Et1v8EQP10U

iii) https://www.youtube.com/watch?v=yNul2STgWyI

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Culture and growth of bacteria

Bacterial growth can be defined as an orderly increase of all the chemical components of the cell. Cell multiplication is a

consequence of growth that leads to an increase in the number of bacteria making up a population or culture. Most bacteria

divide by binary fission in which the bacteria undergo cell division to produce two daughter cells identical to the parent cell.

Bacterial growth can be equated to cell number: one bacterium divides into two, these two produce four, and then eight, and

so on. The growth rate of a bacterium is therefore measured by measuring the change in bacterial number per unit time.

Generation Time

Generation time is the time required for a bacterium to give rise to two daughter cells under optimum conditions. The

generation time for most of the pathogenic bacteria, such as E. coli, is about 20 minutes. The generation time is longer

(i.e.,20 hours) for M. tuberculosis and longest (i.e., 20 days) for M. leprae. A bacterium replicates and multiplies rapidly

producing millions of cells within 24 hours. For example, E. coli in about 7 hours can undergo 20 generations and produce 1

million cells, in about 10 hours undergo 30 generations and produce1 billion cells, and in 24 hours produces 1021 cells

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However, in actual practice, the multiplication of bacteria is arrested after a few cell divisions due to exhaustion of nutrients

and accumulation of toxic products.

Bacterial Growth Curve

When a broth culture is inoculated with a small bacterial inoculum, the population size of the bacteria increases showing a

classical pattern. The bacterial growth curve shows the following four distinct phases

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1. Lag phase: After a liquid culture broth is inoculated, the multiplication of bacteria does not start immediately. It takes

some time to multiply. The time between inoculation and beginning of multiplication is known as lag phase. In this phase,

the inoculated bacteria become acclimatized to the environment, switch on various enzymes, and adjust to the

environmental temperature and atmospheric conditions. During this phase, there is an increase in size of bacteria but no

appreciable increase in number of bacterial cells. The cells are active metabolically. The duration of the lag phase varies

with the bacterial species, nature of culture medium, incubation temperature, etc. It may vary from 1 hour to several days.

2. Log phase: This phase is characterized by rapid exponential cell growth (i.e., 1 to 2 to 4 to 8 and so on). The bacterial

population doubles during every generation. They multiply at their maximum rate. The bacterial cells are small and

uniformly stained. The microbes are sensitive to adverse conditions, such as antibiotics and other antimicrobial agents.

3. Stationary phase: After log phase, the bacterial growth almost stops completely due to lack of essential nutrients, lack of

water oxygen, change in pH of the medium, etc. and accumulation of their own toxic metabolic wastes. Death rate of

bacteria exceeds the rate of replication of bacteria. Endospores start forming during this stage. Bacteria become Gram

variable and show irregular staining. Many bacteria start producing exotoxins.

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4. Decline phase: During this phase, the bacterial population declines due to death of cells. The decline phase starts due to

(a) accumulation of toxic products and autolytic enzymes and (b) exhaustion of nutrients. Involution forms are common in

this stage.

The continuous culture is a method of culture useful for industrial and research purpose. This is achieved by using a special

device for replenishing nutrients and removing bacterial population continuously so that bacteria growth is not inhibited due

to lack of nutrients or due to accumulation of toxic bacterial metabolites.

Nutritional requirements of bacteria

Micronutrients

They are critical for cell function and required in small amounts. They are mostly metals Important in enzymes activity.

Examples include chromium ,cobalt, manganese, molybdenum, nickel, selenium, Tungsten, vanadium, zinc.

Growth factors

These are organic compounds required in small amounts by some cells. Essential for cell metabolism. They include a wide

range of compounds for example amino acids, vitamins,purines, pyrimidines, sterolse.t.c

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Some microbes require preformed growth factors from the environment. Most commonly needed are the vitamins which

function as co-enzymes

Nutritional types of bacteria

The main determinants of a microbes’ nutritional types are its sources of carbon and energy. For instance Autotrophs Use

inorganic carbon ( carbon dioxide) is its primary carbon source while Heterotrophs – are dependent on organic carbon

compounds.

In terms of energy source, microbes that photosynthesize are generally classified as photoptrophs and those that oxidize

chemical compounds are chemotrophs.

Nutritional categories of microbes by carbon and energy source

Category Carbon source Energy example

source
Autotroph Carbon dioxide Non living Photosynthetic organism
environment such as cyanobacteria
Photoautotroph Carbon dioxide Sunlight Cyanobacteria

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 Chemoautotroph Carbondioxide Simple Methanogens
inorganic
chemicals
Heterotrophs Organic Other
organisms or
sunlight
Photoheterotrophs Organic sunlight Non sulfur bacteria
chemoheterotrophs Organic Metabolic Protozoa, fungi. Many
conversion bacteria
of nutrients
from other
organisms
Saprobe Organic Metabolizing Fungi and
the organic bacteria(decomposers)
matter of
dead
organisms
Parasite Organic Utilizing the Various parasites and
tissues pathogens; can be bacteria,
,flluids of a fungi, protozoa, animals
live host

Bacteriological Culture media

Laboratory diagnosis of an infection is usually confirmed by isolating and culturing microorganisms in artificial media.

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Bacteria and fungi are cultured in either liquid (broth) or on solid (agar) artificial media. Koch pioneered the use of agar as a

base for culture media. He developed the pour plate method and was the first to use solid culture media for culture of

bacteria.

At first, Koch cultured bacteria on the sterile surfaces

of cut, boiled potatoes. This was unsatisfactory, because bacteria would not always grow well on potatoes. He then tried to

solidify regular liquid media by adding gelatin. Separate bacterial colonies developed after the surface had been streaked

with a bacterial sample. The sample could also be mixed with liquefied gelatin medium. When the gelatin medium

hardened, individual bacteria produced separate colonies. Despite its advantages, gelatin was not an ideal solidifying agent

because it was digested by many bacteria and melted when the temperature rose above 28°C. A better alternative was

provided by Fannie Eilshemius Hesse, the wife of Walther Hesse, one of Koch’s assistants. She suggested the use of agar as

a solidifying agent— she had been using it successfully to make jellies for sometime. Agar was not attacked by most

bacteria and did not melt until it reaches a temperature of 100°C. One of Koch’s assistants, Richard Petri, developed the

Petri dish (plate), a container for solid culture media.

The basic constituents of culture media include the following:

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Agar

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Agar is the main component that is used universally for preparation of solid media. It is obtained from a variety of sea

weeds and after necessary processing is usually available as powder or as long shreds. Agar is chiefly composed of:

1. A long-chain polysaccharide, consisting of D -galactopyranose

units; A variety of inorganic salts, minute quantities of protein-like materials, traces of long-chain fatty acids; and Minerals,

such as calcium and magnesium. Agar is usually used in a concentration of 2–3%. Agar is hydrolyzed at high temperatures

and at high acid or alkaline pH.

Agar has a unique property of melting at 98°C and solidifying at 42°C.

Peptone

Peptone is another important ingredient of culture media. It is a complex mixture of partially digested proteins. It is

obtained by digestion of lean meat or other protein materials (such as heart muscle, Casein, fibrin, or soya flour) with

proteolytic enzymes (such as pepsin, trypsin, or papain).

Other Ingredients

Other common ingredients of media include water, sodium chloride and other electrolytes, meat extract, yeast extract, malt

extract, blood, and serum. Meat extract is available commercially as Lab-Lemco and contains inorganic salts, carbohydrates,

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certain growth factors, and protein degradation products. Blood or serum is usually used for enriching culture of bacteria.

Usually, 5–10% defibrinated sheep or human blood is used.

Culture media can be classified in several ways:

1.Liquid Media,

2.Semisolid Media,

3.Solid Media

1.Liquid media

Liquid media provide greater sensitivity for the isolation of small numbers of microorganisms. examples of liquid media

include nutrient broth, sugar media, and enrichment media.

Composition and uses of some common liquid media

Media Composition Uses

Peptone water Peptone, sodium chloride, Routine culture,
water base for sugar
fermentation test,
indole test
Nutrient broth Peptone water, meat extract Routine culture

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 Glucose broth Nutrient broth, glucose Blood culture,
culture of fastidious
organisms, such as
streptococci
Brain heart Sodium citrate, sodium Whole blood, bone
infusion broth chloride, sodium phosphate, marrow, body fluid
dextrose peptone, brain and culture
heart infusion broth (ox),
sodium
polyanethol sulfonate (SPS)
Alkaline Peptone water (pH 8.6) Enrichment medium
peptone water for Vibrio
Selenite-F Peptone water, sodium selenite Enrichment medium
broth for feces for
Salmonella and Shigella
Tetrathionate Nutrient broth, sodium Culture of feces for
broth thiosulfate, calcium carbonate, Salmonella
iodine solution
Robertson’s Nutrient broth, predigested Anaerobic bacterial
cooked meat cooked meat of ox heart Culture
(RCM) broth

Liquid media have the following disadvantages:

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a) Identification of mixed cultures growing in liquid media requires subculture onto solid media so that isolated colonies can

be processed separately for identification.

b) Growth in liquid media also cannot ordinarily be quantitated.

c) Bacteria grown in liquid cultures often form colloidal suspensions.

◗ Solid media

Agar is used as a solidifying agent in most culture media. By varying the concentration of agar, it is possible to make the

medium solid or semisolid. Solid media, though somewhat less sensitive than liquid media, provide isolated colonies that

can be quantified and identified. Some genera and species can be recognized on the basis of their colony morphologies.

Media Composition Uses

MacConkey Peptone, lactose, sodium Culture of Gramnegative
medium taurocholate, agar, neutral bacteria, such
red as Escherichia coli
Nutrient agar Nutrient broth, agar 2% Routine culture
Blood agar Nutrient agar, 5% sheep or Routine culture, culture
human blood of fastidious organisms,
such as Streptococcus spp.
Chocolate agar Heated blood agar Culture of Haemophilus

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 influenzae and Neisseria
Deoxycholate Nutrient agar, sodium Culture of Shigella spp.
citrate agar deoxycholate, sodium and Salmonella spp.
citrate, lactose, neutral
red, etc.
Thiosulfate Thiosulfate, citrate, Culture of Vibrio
citrate bile salt bile salt, sucrose, cholerae
sucrose agar bromothymol blue, thymol
blue
Loeffler’s serum Nutrient broth, glucose, Culture of
slope horse serum Corynebacterium
diphtheriae
Lowenstein– Coagulated hen’s egg, Culture of
Jensen medium mineral salt solution, Mycobacterium
asparagine, malachite green tuberculosis

Semisolid media

Addition of reduced concentration of agar (0.2–0.4%) makes the medium semisolid, which facilitates spread of the bacteria

in the medium.

Simple, Complex, Defined, and Special Media

Simple media

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The simple or basal media include nutrient broth and peptone water, which form the basis of other media.

1. Nutrient broth is an example of a simple liquid medium that consists of peptone, meat extract, sodium chloride, and
water.

Addition of 0.5% glucose to nutrient broth makes it glucose broth.

2. Nutrient agar is an example of a simple solid medium. The medium is used routinely for isolation of many bacteria from

clinical specimens.

Complex media

Most of the media other than basal media are usually known as complex media [e.g., chocolate agar, MacConkey agar,

Robertson’s cooked meat (RCM) medium, Lowenstein–Jensen (LJ) medium, etc.]. Complex media have some complex

ingredients, which consist of a mixture of many chemicals in unknown proportions. This is an undefined medium, because

the amino acid source contains a variety of compounds with the exact composition unknown. The complex media contain:

■ Water, A carbon source such as glucose for bacterial growth, Various salts needed for bacterial growth, and source of

amino acids and nitrogen (e.g., beef and yeast extract).

◗ Defined media

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A defined medium, also known as synthetic medium, contains known quantities of all ingredients. All the chemicals used

are known, and it does not contain any animal, yeast, or plant tissue. These media consist of: Trace elements and vitamins;

A defined carbon source and nitrogen source required by certain microbes. Glucose or glycerol is often used as carbon

sources and ammonium salts or nitrates as inorganic nitrogen sources.

◗ Special media

These include (a) enriched media, (b) enrichment media, (c) selective media, (d) indicator or differential media, (e)

transport media, and ( f ) sugar media.

Enriched media: The enriched media are invariably solid media that facilitate growth of certain fastidious bacteria. These

media are prepared by adding substances like blood, serum, and egg to the basal media in order to meet the nutritional

requirements of more exacting and more fastidious bacteria. Blood agar(Color Photo 1), chocolate agar, Loeffler’s serum

slope (LSS), and LJ medium are some examples of enriched media. Blood agar is an enriched medium in which

nutritionally rich whole blood supplements constitute the basic nutrients. Chocolate agar is enriched with heat-treated blood

(80°C), which turns brown and gives the medium the color for which it is named.

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Enrichment media: Enrichment media are liquid media that stimulate the growth of certain bacteria or suppress the growth

of others for isolation of desired pathogenic bacteria. Commensal bacteria, such as Escherichia coli present in feces, tend to

overgrow pathogenic ones in stool specimen. In such

situations, enrichment media (such as selenite-F broth or tetrathionate broth) are used for the isolation of Salmonella Typhi

and Shigella spp. from feces.

NB. Enrichment media are useful for isolation of wanted bacteria from stool and other specimens containing more than one

species of bacteria.

Selective media: These are solid media that contain substances that inhibit the growth of all but a few bacteria but at the

same time facilitate isolation of certain bacteria. Some examples of selective media include:

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■ Thiosulfate citrate bile salt sucrose agar (TCBS)

(Color Photo 35) selective for the isolation of Vibrio

cholerae.

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Deoxycholate citrate agar (DCA) selective for enteric bacilli,

such as Salmonella spp. and Shigella spp.

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LJ medium selective for Mycobacterium tuberculosis.

Differential or indicator media: Differential or indicator media distinguish one microorganism from another growing on

the same media by their growth characteristics. NB. Differential or indicator media depend on the biochemical properties

of a
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microorganism growing in the presence of specific nutrients or indicators, such as neutral red, phenol red, eosin, or

methylene blue. The medium changes color when a bacterium grows in them. For example,. Lactose fermenting bacteria,

such as E. coli produce pink colonies

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(Color Photo 2), whereas nonlactose fermenting bacteria, such as Salmonella spp. form pale or colorless colonies on

MacConkey agar. Fermentation of lactose in the medium makes it acidic and leads to the formation of pink colonies in the

presence of neutral red.

Examples of differential media include:

■ Eosin methylene blue (EMB), differential for lactose and sucrose fermentation;

Some selective media used in routine microbiology laboratories

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Medium Colony characteristics Colony characteristics Organism inhibited
Mannitol salt Big yellow Small pink colonies Streptococcus
agar colonies of of Staphylococcus spp.
Staphylococcus epidermidis
Aureus
Thayer– Gray colonies Gram-positive
Martin of Neisseria Cocci
medium meningitidis
and Neisseria
gonorrhoeae
MacConkey Lactose Lactose Gram-positive
agar medium fermenters: red nonfermenters: Cocci
colonies, e.g., colorless colonies of
Escherichia coli Salmonella spp.
Thiosulfate Sucrose Sucrose Enteric bacilli
citrate bile fermenter: nonfermenters:
salt sucrose yellow colonies green colonies
agar of Vibrio cholerae of Vibrio
parahaemolyticus
Charcoal Cut glass Gram-positive
yeast extract colonies of cocci
Legionella spp.
Lowenstein– Rough, tough, Smooth and Cocci
Jensen and buff pigmented
medium colonies of colonies of atypical
Mycobacterium Mycobacterium spp.
tuberculosis

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Transport media:

Transport media are used to maintain the viability of certain delicate organisms in clinical specimens during their transport

to the laboratory. They typically contain only buffers and salt. They lack carbon, nitrogen, and organic growth factors,

hence do not facilitate microbial multiplication. Examples of transport media are Stuart’s transport medium for Neisseria

gonorrhoeae. Factors Affecting Growth of Bacteria

1. Oxygen
Bacteria on the basis of their oxygen requirements can be classified broadly into aerobic and anaerobic bacteria.
Aerobic bacteria: They require oxygen for their growth. They may be:
1a) Obligate aerobes—which can grow only in the presence of oxygen e.g Pseudomonas. aeruginosa).
b) Facultative aerobes—which are ordinary aerobes but can also grow without oxygen (e.g., E. coli). Most of the pathogenic
bacteria are facultative aerobes. c) Microaerophilic bacteria—those bacteria that can grow in the presence of low oxygen
and in the presence of low (4%) concentration of carbon dioxide (e.g., Campylobacter jejuni).
d) Aerotolerant Some fermentative organisms (e.g., Lactobacillus plantarum) are but do not contain the enzyme catalase or
superoxide dismutase. Oxygen is not reduced, and therefore hydrogen peroxide (H2O2) and nascent oxygen (O2 _) are not
produced.

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Anaerobic bacteria: Obligate anaerobes are the bacteria that can grow only in the absence of oxygen (e.g., Clostridium
botulinum Clostridium tetani, etc.). These bacteria lack superoxide dismutase and catalase; hence oxygen is lethal to these
organisms.

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2
◗ Carbon dioxide: The organisms that require higher amounts of carbon dioxide
(CO2) for their growth are called capnophilic bacteria. They grow well in the presence of 5–10% CO2 and 15% O2. In
candle jar, 3% CO2 can be achieved. Examples of such bacteria include H. influenzae, Brucella abortus, etc.

◗ Temperature

The optimum temperature for most of the pathogenic bacteria is 37oC. The optimal temperature, however, is variable;

depending on their temperature range, growth of bacteria is grouped as follows:

a) Psychrophiles: These bacteria are cold loving microbes that grow within a temperature range of 0–20 oC. Most of soil and

water saprophytes belong to this group.

c)Mesophiles: These are moderate temperature loving microbes that grow between 25 oC and 40 oC. Most of pathogenic

bacteria belong to this group.

c)Thermophiles: These are heat loving microbes. They can grow at a high temperature range of 55–80_C. B.

stearothermophilus is an example.

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pH

Most pathogenic bacteria grow between pH 7.2 and 7.6. Very few bacteria, such as lactobacilli, can grow at acidic pH below

4.0. Many food items, such as pickles and cheese, are prevented from spoilage by acids produced during fermentation. V.

cholerae is an example of the bacteria that can grow at an alkaline (8.2–8.9) pH.

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The enzymes in the cell membrane are sensitive to the Hydrogen concentration

◗ Light

Depending on the source of energy bacteria make use of, they may be classified as phototrophs (bacteria deriving energy

from sunlight) or chemotrophs (bacteria deriving energy from chemical sources).

◗ Osmotic pressure

Microbes obtain almost all their nutrients in solution from surrounding water. Hence factors such as osmotic pressure and

salt concentration of the solution affect the growth of bacteria. Bacteria by virtue of mechanical strength of their cell wall

are able to withstand a wide range of external osmotic variations. Organisms requiring high osmotic pressures are called

osmophilic bacteria. Sudden exposure of bacteria to hypertonic solution may cause osmotic withdrawal of water, leading to

osmotic shrinkage of the protoplasm ( plasmolysis). On the other hand sudden transfer of bacteria from concentrated

solution to distilled water may cause excessive imbibition of water leading to

swelling and bursting of cell ( plasmoptysis).

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Enumeration of microorganisms

For unicellular microorganisms, such as bacteria, the reproduction of the cell reproduces the entire organism. Therefore,

microbial growth is essentially synonymous with microbial reproduction. To determine rates of microbial growth and death,

it is necessary to enumerate microorganisms, that is, to determine their numbers. It is also often essential to determine the

number of microorganisms in a given sample. For example, the ability to determine the safety of many foods and drugs

depends on knowing the levels of microorganisms in those products. A variety of methods has been developed for the

enumeration of microbes. These methods measure cell numbers, cell mass, or cell constituents that are proportional to cell

number. The four general approaches used for estimating the sizes of microbial populations are direct and indirect counts of

cells and direct and indirect measurements of microbial biomass. Each method will be described in more detail below.

1. Direct Count of Cells Cells are counted directly under the microscope or by an electronic particle counter. Two of the

most common procedures used in microbiology are discussed below.

a) Direct Count Using a Counting Chamber Direct microscopic counts are performed by spreading a measured volume

of sample over a known area of a slide, counting representative microscopic fields, and relating the averages back to

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 the appropriate volume-area factors. Specially constructed counting chambers, such as the Petroff-Hauser and Levy

counting chambers, simplify the direct counting procedure because they are made with depressions in which a known

volume overlies an area that is ruled into squares. The ability to count a defined area and convert the numbers

observed directly to volume makes the direct enumeration procedure relatively easy. Direct counting procedures are

rapid but have the disadvantage that they do not discriminate between living and dead cells. This method is used to

assess the sanitation level of a food product and in performing blood cell counts in hematology. The differential white

blood cell count, which is used as an indication of the nature of a microbial infection, involves direct counting of

blood cells that have been stained to differentiate different types of white blood cells.

b) Direct Count Using Fluorescent Dyes

Fluorescent dyes are becoming more used in recent years for a variety of procedures, one of which is bacterial counts.

These dyes can be employed to stain all species, a particular species of interest in an environmental sample or even a

specific component of cells.

The most widely used fluorescent dye for counting the number of bacterial cells is acridine orange which stains both

living and dead cells by interacting with DNA and protein components of cells. The stained cells fluoresce orange

when
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excited near ultraviolet light. This stain is particularly useful for determining the total number of microorganisms in

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 samples, such as soil and water, where the co-existence of metabolically diverse populations precludes establishing

conditions for the simultaneous enumeration of microbial populations by viable count procedures. The procedure is

widely used in marine microbiology where population levels are often low and where viable plate counts are known

to severely underestimate total number of bacteria.

Typically, the viable count is less than 1% of the direct count for marine samples. In this procedure the bacteria in a

known volume of sample are stained with acridine orange and the sample is then filtered through a 0.22 μm filter. The

bacteria are trapped on the filter that is then examined under a fluorescence microscope. The bacteria in a defined

area of the filter are counted and the concentration in the original sample is then calculated. Other fluorescent dyes

that are also gaining popularity are cyanoditolyl tetrazolium chloride (CTC), auramine and rhodamine. CTC binds to

respiration proteins in the cell and thus can demonstrate live cells. Auramine and rhodamine bind to cell wall of

Mycobacteria and emit bright yellow or orange color under a fluorescent microscope.

c) 2. Indirect Count of Cells

Microorganisms in a sample are diluted or concentrated and grown on a suitable medium; the development of
growing microorganisms (for example, colony formation on agar plates) is then used to estimate the numbers of
microorganisms in the original
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 sample.

Viable Count
The most common procedure for the enumeration of bacteria is the viable plate count. In this method, serial dilutions of a

sample containing viable microorganisms are plated onto a suitable growth medium. The suspension is either spread onto

the surface of agar plates (spread plate method), or is mixed with molten agar, poured into plates, and allowed to solidify

(pour plate method). The plates are then incubated under conditions that permit microbial reproduction so that colonies

develop that can be seen without the aid of a microscope. It is assumed that each bacterial colony arises from an individual

cell that has undergone cell division. Therefore, by counting the number of colonies and accounting for the dilution factor,

the number of bacteria in the original sample can be determined.

There are several drawbacks to the viable count method. The major disadvantage is that it is selective and therefore biased.

The nature of the growth conditions, including the composition and pH of the medium used as well as the conditions such as

temperature, determines which bacteria in a mixed population can grow. Since there is no universal set of conditions that

permits the growth of all microorganisms, it is impossible to enumerate all microorganisms by viable plating. This same

disadvantage, however, becomes advantageous when one is interested in only a specific microbial population. For example,

we can design selective procedures for the enumeration of coliforms and other physiologically defined microbial groups.
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The viable count is an estimate of the number of cells. Because some organisms exist as pairs or groups and because mixing

and shaking of the sample does not always separate all the cells, we actually get a count of the "colony forming units". One

cell or group of cells will produce one colony, therefore when we record results for a viable count, it is customary to record

the results as colony forming units per ml (cfu/ml) or per gram (cfu/g) of test material.

Because we generally have no idea of how many bacteria are in a sample, it is almost always necessary to prepare a dilution

series to ensure that we obtain a dilution containing a reasonable number of bacteria to count. Dilutions in the range 10-1

(1/10) to 10-8 (1/100,000,000) are generally used, although with particular types of samples the range of dilutions can be

restricted. For example, for water that is not turbid, the maximal dilution needed is 10-6 because we know that if there were

107 or more bacteria per milliliter, the water would be turbid.

The Most Probable Number (MPN)

The MPN procedure is a statistical method based upon the probability theory.

Samples are serially diluted to the point of extinction, that is, to a point where there are no more viable microorganisms. To

detect the end point, multiple serial dilutions are inoculated into a suitable growth medium, and the development of some

recognizable

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characteristic, such as acid production or turbidity, is used to indicate growth (the presence of at least one viable

microorganism in the diluted sample). The pattern of positive tests (growth) in the replicates and statistical probability

tables are used to determine the concentration (most probable number) of bacteria in the original sample. Statistical MPN

tables are available for replicates of 3, 5, and 10 tubes of each dilution.

The more replicate tubes used, the greater the precision of the estimate of the size of the bacterial population.

3. Direct Measurement of Microbial Biomass

Cell mass is determined directly by weighing whole cells; biomass can be correlated with cell numbers by reference to a

standard curve. Wet weight or dry weight of bacteria may be used for estimation of cell numbers.

4. Indirect Measurement of Microbial Biomass

Microbial biomass is estimated by measuring relatively constant biochemical components of microbial cells, such as

protein, ATP, lipopolysaccharides, peptidoglycan, and chlorophyll; biomass can also be indirectly estimated by measured

turbidity that can

then be correlated with cell numbers by reference to a standard curve. Various procedures based on the detection of specific

microbial macromolecules or metabolic products can be used to estimate numbers of microorganisms. For example,

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peptidoglycan can be quantified, and because this biochemical occurs exclusively in the cell wall of bacteria, the

concentration of peptidoglycan can be used to

estimate bacterial numbers. Such biochemical approaches for determining bacterial numbers depend on the development of

analytical chemical procedures for quantifying the particular biochemical and determining what proportion of bacterial cell

is composed

of the specific biochemical constituent.

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Bacterial Ecology

Prokaryote habitats and functions

Bacteria are ubiquitous and can be found anywhere on our planet. Prokaryotes are also abundant on and within human body.

Bacteria thrive on the human mouth, nasal cavity, throat, ears gastro intestinal tract and vagina. Large colonies of bacteria

can be found healthy human skin especially moist areas. However even drier areas of the skin are not free from bacteria.

Role of bacteria in the environment.

1. They are necessary part of soil formation and stabilization process through breakdown of organic matter and

development of biofilms.

2. Many species of bacteria use substances released from plant roots, such as acids and carbohydrates as nutrients. The

bacteria metabolize these plant substances and release the products of bacterial metabolism back to the soil, forming

humus and thus increasing the soil fertility. for example in salty lakes such as dead sea salt loving halobacteria

decompose dead shrimp and nourish young shrimp and flies with the products of bacterial metabolism.

3. Bacteria are also abundant in air, there may be upto 2000 different kinds of bacteria in the air, similar to their diversity in
the soil. Prokaryotes can be found everywhere on earth because they are extremely resilient and adaptable. They are
often metabolically flexible, which means that they might easily switch from one energy source to another, depending
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on the

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 availability of the sources, or from one metabolic pathway to another. For example Ground water bacteria store complex
high-energy carbohydrates when grown in pure groundwater, but they metabolize these molecules when the groundwater
is enriched with phosphates. Some bacteria get their energy by reducing sulfates into sulfides, but can switch to a
different metabolic pathway when necessary, producing acids and free hydrogen ions. Prokaryotes perform functions
vital to life on earth by capturing (or “fixing”) and recycling elements like carbon and nitrogen.
4. Carbon fixation- Organisms such as animals require organic carbon to grow, but, unlike prokaryotes, they are unable to

use inorganic carbon sources like carbon dioxide. Thus, animals rely on prokaryotes to convert carbon dioxide into

organic carbon products that they can use. This process of converting carbon dioxide to organic carbon products is called

carbon fixation.

5. Plants and animals also rely heavily on prokaryotes for nitrogen fixation, the conversion of atmospheric nitrogen into
ammonia, a compound that some plants can use to form many different biomolecules necessary to their survival (Figure
4.3). Bacteria in the genus Rhizobium, for example, are nitrogen-fixing bacteria; they live in the roots of legume plants
such as clover, alfalfa, and peas. Ammonia produced by Rhizobium helps these plants to survive by enabling them to
make building blocks of nucleic acids. In turn, these plants may be eaten by animals—sustaining their growth and
survival—or they may die, in which case the products of nitrogen fixation will enrich the soil and be used by other
plants.

42
6. Prokaryotes play a role in cleaning up the environment. Recently, some researchers focused on the diversity and

functions of prokaryotes in man made environments. They found that some bacteria play a unique role in degrading toxic

chemicals that pollute water and soil

7. Some bacteria are human pathogens that may cause illness or infection when they enter the body. In addition, some

bacteria can contaminate food, causing spoilage or foodborne illness, which makes them subjects of concern in food

preparation and safety. Less than 1% of prokaryotes are thought to be human pathogens.

8. Prokaryotes are now thought to be key players in the processes of climate change. In recent years, as temperatures in the

earth’s polar regions have risen, soil that was formerly frozen year-round (permafrost) has begun to thaw. Carbon

trapped in the permafrostis gradually released and metabolized by prokaryotes. This produces massive amounts of

carbon dioxide and methane, greenhouse gases that escape into the atmosphere and contribute to the greenhouse effect.

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 Symbiotic Relationships

Prokaryotic microorganisms can associate with plants and animals. Often, this association results in unique relationships

between organisms. For example, bacteria living on the roots or leaves of a plant get nutrients from the plant and, in return,

produce substances that protect the plant from pathogens.

On the other hand, some bacteria are plant pathogens that use mechanisms of infection similar to bacterial pathogens of

animals and humans.

Bacteria live in a community, or a group of interacting populations of organisms.

A population is a group of individual organisms belonging to the same biological species and limited to a certain

geographic area.

 Populations can have cooperative interactions, which benefit the populations,

 Competitive interactions, in which one population competes with another for resources.

The study of these interactions between bacteria populations is called bacteria ecology. Any interaction between

different species within a community is called symbiosis. Such interactions fall along a continuum between opposition

44
 and cooperation. Interactions in a symbiotic relationship may be beneficial or harmful, or have no effect on one or both

of the species involved.

The main types of symbiotic interactions among prokaryotes.

Type Population A Population B

Mutualism Benefitted Benefitted

Commensalism Benefitted Unaffected

Amensalism Harmed Unaffected

Neutralism Unaffected Unaffected

Parasitism Benefitted Harmed

45
 1. Mutualism / Syntropy/ Crossfeeding

When two species benefit from each other, the symbiosis is called mutualism (or syntropy, or crossfeeding). For example,

humans have a mutualistic relationship with the bacterium Bacteroides thetaiotetraiotamicron, which lives in the intestinal

tract. B. thetaiotetraiotamicron digests complex polysaccharide plant materials that human digestive enzymes cannot break

down, converting them into monosaccharides that can be absorbed by human cells. Humans also have a mutualistic

relationship with certain strains of Escherichia coli, another bacterium found in the gut. E. coli relies on intestinal contents

for nutrients, and humans derive certain vitamins B12 from E. coli, particularly vitamin K,which is required for the

formation of blood clotting factors. (This is only true for some strains of E.coli, however. Other strains are pathogenic and

do not have a mutualistic relationship with humans.)

2. Amensalism

A type of symbiosis in which one population harms another but remains unaffected itself is called amensalism. In the case

of bacteria, some amensalist species produce bactericidal substances that kill other species of bacteria. For example, the

46
bacterium Lucilia sericata produces a protein that destroys Staphylococcus aureus, a bacterium commonly found on the

surface of the human skin. Too much handwashing can affect this relationship and lead to S. Aureus diseases and

transmission.

3. Commensalism

Commensalism is where one organism benefits while the other is unaffected. This occurs when the bacterium

Staphylococcus epidermidis uses the dead cells of the human skin as nutrients. Billions of these bacteria live on our skin,

but in most cases (especially when our immune system is healthy), we do not react to them in any way

4. Neutralism. An example of neutralism is the coexistence of metabolically active (vegetating) bacteria and

endospores (dormant,metabolically passive bacteria). For example, the bacterium Bacillus anthracis typically forms

endospores in soil when conditions are unfavorable. If the soil is warmed and enriched with nutrients, some

endospores germinate and remain in symbiosis with other endospores that have not germinated.

5. Parasitism. The relationship between humans and many pathogenic prokaryotes can be characterized as parasitic

because these organisms invade the body, producing toxic substances or infectious diseases that cause harm. Diseases

such as tetanus, diphtheria, pertussis, tuberculosis, and leprosy all arise from interactions between bacteria and

47
humans. Scientists have coined the term microbiome to refer to all prokaryotic and eukaryotic microorganisms that

are associated

48
with a certain organism. Within the human microbiome, there are resident microbiota and transient microbiota. The

resident microbiota consists of microorganisms that constantly live in or on our bodies.

The term transient microbiota refers to microorganisms that are only temporarily found in the human body, and

these may include pathogenic microorganisms. Hygiene and diet can alter both the resident and transient microbiota.

The Resident microbiota (consistently found in certain environmental niche) is amazingly diverse, not only in terms

of the variety of species but also in terms of the preference of different microorganisms for different areas of the

human body. For example, in the human mouth, there are thousands of commensal or mutualistic species of bacteria.

Some of these bacteria prefer to inhabit the surface of the tongue, whereas others prefer the internal surface of the

cheeks, and yet others prefer the front or back teeth or gums.The inner surface of the cheek has the least diverse

microbiota because of its exposure to oxygen. By contrast, the crypts of the tongue and the spaces between teeth are

two sites with limited oxygen exposure, so these sites have more diverse microbiota, including bacteria living in the

absence of oxygen (e.g.,Bacteroides, Fusobacterium). Differences in the oral microbiota between randomly chosen

human individuals are

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 also significant. Studies have shown, for example, that the prevalence of such bacteria as Streptococcus, Haemophilus,

Neisseria, and others was dramatically different when compared between individuals.

There are also significant differences between the microbiota of different sites of the same human body. The inner surface

of the cheek has a predominance of Streptococcus, whereas in the throat, the palatine tonsil, and saliva, there are two to

three times fewer Streptococcus, and several times more Fusobacterium. In the plaque removed from gums, the predominant

bacteria belong to the genus Fusobacterium. However, in the intestine, both Streptococcus and Fusobacteriumdisappear, and

the genus Bacteroides becomes predominant. Notonlycan the microbiota vary from one body site to another, the

microbiome can also change over time within the same individual. Humans acquire their first inoculations of normal flora

during natural birth and shortly after birth. Before birth, there is a rapid increase in the population of Lactobacilli spp. in the

vagina, and this population serves as the first colonization of microbiota during natural birth. After birth, additional

microbes are acquired from healthcare providers, parents, other relatives, and individuals who come in contact with the

baby. This process establishes a microbiome that will continue to evolve over the course of the individual’s life as new

microbes colonize and are eliminated

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from the body. For example,it is estimated that within a 9 hour period, the microbiota of the small intestine can change so

that half of the microbial inhabitants will be different.

The importance of the initial Lactobacilli colonization during vaginal child birth is highlighted by studies demonstrating a

higher incidence of diseases in individuals born by cesarean section, compared to those born vaginally. Studies have shown

that babies born vaginally are predominantly colonized by vaginal Lactobacilli, whereas babies born by cesarean section are

more frequently colonized by microbes of the normal skin microbiota, including common hospital-acquired pathogens.

Throughout the body, resident microbiotas are important for human health because they occupy niches that might be

otherwise taken by pathogenic microorganisms. For instance, Lactobacilli spp. are the dominant bacterial species of the

normal vaginal microbiota for most women. Lactobacilli produce lactic acid, contributing to the acidity of the vagina and

inhibiting the growth of pathogenic yeasts. However, when the population of the resident microbiota is decreased for some

reason (e.g., because of taking antibiotics), the pH of the vagina increases, making it a more favorable environment for the

growth of yeasts such as Candida albicans. Antibiotic therapy can also disrupt the microbiota of the intestinal tract and

respiratory tract, increasing the risk for secondary infections and/or promoting the long-term carriage and shedding of

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pathogens

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Bacterial genetics

Genomic DNA contains structural genes which encode products that serve as cellular structures or enzymes. They

also contain Regulatory genes which encode products that regulate gene expression. The Expression of a gene is a

highly regulated process. In bacteria the aim is gene expression is to ensure that a cells’ resources are not wasted

making proteins that the cells does not need at that time.

LAC OPERON CONCEPT

Bacteria utilize a special energy saving system of genetic control called operon. The operon is a sequence of DNA

that contains multiple genes used to produce multiple proteins for a single purpose. An example of an operon is the

lac operon in Escherichia coli. In order to break down lactose, E. Coli must use a series of enzymes:

Betagalactosidase, Galactosidase, Transacetylase

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 The genes for the 3 enzymes are located in a row on a DNA and share a single promoter. Genes determining

structure of a particular protein are called structural genes and the activity of the structural genes are controlled by

regulator genes which lie adjacent to them.

The gens lac Z, lac Y and Lac A code are structural genes that code for the three enzymes.

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Lac I gens code for the repressor protein, hence is the regulator gene.

Between the LacI gene and structural gene lie promoter and operator gens. During transcription of the structural gene,

the enzyme RNA polymerase binds to promoter region and goes through the operator region. Under normal

circumstances, when the structural genes are not transcribed, the repressor protein is bound to the operator region thus

preventing the passage of RNA polymerase from the operator region towards the operon.

When lactose is available in the environment, the repressor protein in the operator region binds to lactose because it

has a high affinity for lactose. This frees the operator region and the RNA polymerase enzyme moves towards the

operon and transcribes the structural gene. The products of structural genes result in the metabolism of lactose. When

lactose is no more available, the repressor protein goes back and binds to the operator region stopping further

transcription of structural gene.

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 MAJOR BACTERIAL GROUPS

PHYLUM PROTEOBACTERIA

These are gram negative bacteria. It includes many bacteria that are part of the normal human microbiota as well as many

pathogens. The Proteobacteria are further divided into five classes:

a) Alphaproteobacteria,

b) Betaproteobacteria

c) Gammaproteobacteria,

d) Deltaproteobacteria

e)Epsilonproteobacteria

Alphaproteobacteria

The first class of Proteobacteria is the Alphaproteobacteria. The unifying characteristic of this class is that they are

oligotrophs, organisms capable of living in low-nutrient environments such as deep oceanic sediments, glacial ice, or

deep undersurface soil.

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Among the Alphaproteobacteria are two taxa, chlamydias and rickettsias, that are obligate intracellular pathogens,

meaning that part of their life cycle must occur inside other cells called host cells. When not growing inside a host

cell, Chlamydia and Rickettsia are metabolically inactive outside of the host cell. They cannot synthesize their own

adenosine triphosphate (ATP), and, therefore, rely on cells for their energy needs. Rickettsia spp. include a number of

serious human pathogens. For example, R. rickettsii causes Rocky Mountain spotted fever, a life-threatening form of

meningoencephalitis (inflammation of the membranes that wrap the brain). R. rickettsii infects ticks and can be transmitted

to humans via a bite from an infected tick Another species of Rickettsia, R. prowazekii, is spread by lice. It causes epidemic

typhus, a severe infectious disease common during warfare and mass migrations of people. R. prowazekii infects human

endothelium cells, causing inflammation of the inner lining of blood vessels, high fever, abdominal pain, and sometimes

delirium.

Chlamydia is another taxon of the Alphaproteobacteria. Members of this genus are extremely resistant to the cellular

defenses, giving them the ability to spread from host to host rapidly via elementary bodies. The metabolically and

reproductively inactive elementary bodies are the endospore-like form of intracellular bacteria that enter an epithelial

cell, where they become active

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C. trachomatis is a human pathogen that causes trachoma, a disease of the eyes, often leading to blindness. C.

trachomatis also causes the sexually transmitted disease lymphogranuloma venereum (LGV). This disease is often

mildly symptomatic, manifesting as regional lymph node swelling, or it may be asymptomatic, but it is extremely

contagious and is common on college campuses.

Table showing alphaproteobacteria

Genus Morphology Unique characterisitics

Brucella Gram negative flagellated coccibacilli Facultative intracellular bacteria, transmitted by contaminated
milk from infected cows, cause brucellosis in cattle and
humans

Chlamydia Gram-negative, coccoid Obligatory intracellular bacteria; some cause chlamydia,

trachoma, and pneumonia

Rhizobium Gram-negative, Nitrogen-fixing bacteria that live in soil and form symbiotic
rectangular bacilli with relationship with roots of legumes (e.g., clover, alfalfa, and
rounded ends forming beans)
clusters

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 Rickettsia Gram-negative, highly Obligate intracellular bacteria; transmitted by ticks; may cause
pleomorphic bacteria Rocky Mountain spotted fever and typhus
(may be cocci, rods, or
threads)

Agrobacterium Gram negative bacilli Plant pathogen; one species, A. tumefaciens, causes tumors in
plants

Betaproteobacteria

the class Betaproteobacteria are eutrophs (or copiotrophs), meaning that they require a copious amount of organic

nutrients. Betaproteobacteria often grow between aerobic and anaerobic areas (e.g., in mammalian intestines). Some genera

include species that are human pathogens, able to cause severe, sometimes life-threatening disease. The genus Neisseria, for

example, includes the bacteria N. gonorrhoeae, the causative agent of the STI gonorrhea, and N. meningitides, the causative

agent of bacterial meningitis. Neisseria are cocci that live on mucosal surfaces of the human body. They are fastidious, or

difficult to culture, and they require high levels of moisture, nutrient supplements, and carbon dioxide. Also, Neisseria are

microaerophilic,meaning that they require low levels of oxygen. For optimal growth and for the purposes of identification,

Neisseria

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spp. are grown on chocolate agar (i.e., agar supplemented by partially hemolyzed red blood cells). Their characteristic

pattern of growth in culture is diplococcal: pairs of cells resembling coffee beans.

Culture of Neisseria

The pathogen responsible for pertussis (whooping cough) is also a member of Betaproteobacteria. The bacterium

Bordetella pertussis, from the order Burkholderiales, produces several toxins that paralyze the movement of cilia in

60
the human respiratory tract and directly damage cells of the respiratory tract, causing a severe cough.

Genus Morphology Unique characteristics

Bordetella A small, gram-negative Aerobic, very fastidious; B. pertussis causes pertussis (whooping
coccobacillus cough)

Burkholderia Gram-negative bacillus Aerobic, aquatic, cause diseases in horses and humans (especially
patients with cystic fibrosis); agents of nosocomial infections

Leptothrix Gram-negative, Aquatic; oxidize iron and manganese; can live in wastewater
sheathed, filamentous treatment plants and clog pipes
bacillus

Neisseria Gram-negative, coffee Require moisture and high concentration of carbon dioxide; oxidase
bean-shaped coccus positive, grow on chocolate agar; pathogenic species cause
forming pairs gonorrhea and meningitis

Thiobacillus Gram-negative bacillus Thermophilic, acidophilic, strictly aerobic bacteria; oxidize iron and
sulfur

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Gammaproteobacteria

The most diverse class of gram-negative bacteria is Gammaproteobacteria, and it includes a number of human

pathogens. For example, a large and diverse family, Pseudomonaceae, includes the genus Pseudomonas. Within this

genus is the species P. aeruginosa, a pathogen responsible for diverse infections in various regions of the body. P.

aeruginosa is a strictly aerobic, nonfermenting, highly motile bacterium. It often infects wounds and burns, can be

the cause of chronic urinary tract infections, and can be an important cause of respiratory infections in patients with

cystic fibrosis or patients on mechanical ventilators. Infections by P. aeruginosa are often difficult to treat because

the bacterium is resistant to many antibiotics and has a remarkable ability to form biofilms.

Other representatives of Pseudomonas include the fluorescent (glowing) bacterium P. fluorescens and the soil bacteria P.

putida, which is known for its ability to degrade xenobiotics (substances not naturally produced or found in living

organisms). The Pasteurellaceae also includes several clinically relevant genera and species. This family includes several

bacteria that are human and/or animal pathogens. For example, Pasteurella haemolytica causes severe pneumonia in sheep

and goats. P. multocida is a species that can be transmitted from animals to humans through bites, causing infections

of the skin and deeper tissues.

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The genus Haemophilus contains two human pathogens, H. influenzae and H. ducreyi. Despite its name, H. influenzae does

not cause influenza (which is a viral disease). H. influenzae can cause both upper and lower respiratory tract infections,

including sinusitis, bronchitis, ear infections, and pneumonia. Before the development of effective vaccination, strains of H.

influenzae were a leading cause of more invasive diseases, like meningitis in children. H. ducreyi causes the STI known as

chancroid.

The order Vibrionales includes the human pathogen Vibrio cholerae. This comma-shaped aquatic bacterium thrives

in highly alkaline environments like shallow lagoons and sea ports. A toxin produced by V. cholerae causes hypersecretion

of electrolytes and water in the large intestine, leading to profuse watery diarrhea and dehydration.

V. parahaemolyticus is also a cause of gastrointestinal disease in humans, whereas V. vulnificus causes serious and

potentially life-threatening cellulitis (infection of the skin and deeper tissues) and blood-borne infections.

Another representative of Vibrionales, Aliivibrio fischeri, engages in a symbiotic relationship with squid. The squid

provides nutrients for the bacteria to grow and the bacteria produce bioluminescence that protects the squid from

predators.The genus Legionella also belongs to the Gammaproteobacteria. L. pneumophila, the pathogen responsible for

Legionnaires disease, is an aquatic bacterium that tends to inhabit pools of warm water, such as those found in the tanks of

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air conditioning units in

64
large buildings. This bacteria can spread in aerosols, outbreaks of Legionnaires disease often affect residents of a building

in which the water has become contaminated with Legionella.

Enterobacteriaceae is a large family of enteric (intestinal) bacteria belonging to the Gammaproteobacteria. They are

facultative anaerobes and are able to ferment carbohydrates. Within this family, microbiologists recognize two distinct

categories. The first category is called the coliforms, after its prototypical bacterium species, Escherichia coli.Coliforms are

able to ferment lactose completely (i.e., with the production of acid and gas). The second category, noncoliforms, either

cannot ferment lactose or can only ferment it incompletely (producing either acid or gas, but not both). The noncoliforms

include some notable human pathogens, such as Salmonella spp., Shigella spp., and Yersinia pestis.

Many strains of E. coli are in mutualistic relationships with humans. However, some strains produce a potentially deadly

toxin called Shiga toxin, which perforates cellular membranes in the large intestine, causing bloody diarrhea and peritonitis

(inflammation of the inner linings of the abdominal cavity). Other E. coli strains may cause traveler’s diarrhea, a less severe

but very widespread disease.

The genus Salmonella, which belongs to the noncoliform group of Enterobacteriaceae, is interesting in that there is still no

consensus about how many species it includes. Scientists have reclassified many of the groups they once thought to be

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species

66
as serotypes (also called serovars), which are strains or variations of the same species of bacteria. Their classification is

based on patterns of reactivity by animal antisera against molecules on the surface of the bacterial cells. A number of

serotypes of Salmonella can cause salmonellosis, characterized by inflammation of the small and the large intestine,

accompanied by fever, vomiting, and diarrhea. The species S. enterobacterica (serovar typhi) causes typhoid fever, with

symptoms including fever, abdominal pain, and skin rashes.

Genus Morphology Unique characteristics

Escherichia Gram-negative Facultative anaerobe; inhabit the gastrointestinal tract of

warmblooded
animals; some strains are mutualists, producing vitamin K;
others, like serotype E. coli O157:H7, are pathogens; E. coli has
been a model organism for many studies in genetics and
molecular
biology

Enterobacter Gram-negative Facultative anaerobe; cause urinary and respiratory tract

infections
bacillus in hospitalized patients; implicated in the pathogenesis of
obesity
Vibrio Gram negative Inhabit seawater; flagellated, motile; may produce toxin that
causes
hypersecretion of water and electrolytes in the gastrointestinal
tract;
some species may cause serious wound infections

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 Pseudomonas Gram-negative Aerobic; versatile; produce yellow and blue pigments, making
them
bacillus appear green in culture; opportunistic, antibiotic-resistant
pathogens may cause wound infections, hospital-acquired
infections, and secondary infections in patients with cystic

fibrosis

Deltaproteobacteria

The Deltaproteobacteria is a small class of gram-negative Proteobacteria that includes sulfate-reducing bacteria (SRBs), so

named because they use sulfate as the final electron acceptor in the electron transport chain. Few SRBs are pathogenic.

However, the SRB Desulfovibrio orale is associated with periodontal disease (disease of the gums). Deltaproteobacteria

also includes the genus Bdellovibrio, species of which are parasites of other gram-negative bacteria. Bdellovibrio invades

the cells of the host bacterium, positioning itself in the periplasm, the space between the plasma membrane and the cell wall,

feeding on the host’s proteins and polysaccharides. The infection is lethal for the host cells. Another type of

Deltaproteobacteria, myxobacteria, lives in the soil, scavenging inorganic compounds. Motile and highly social, they

interact with other bacteria

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within and outside their own group. They can form multicellular, macroscopic “fruiting bodies”. These bacteria can also

form metabolically inactive myxospores.

Genus morphology Unique characteristics

Bdellovibrio Gram-negative, Obligate aerobes; motile; parasitic (infecting

commashaped other
Rod bacteria)

Desulfovibrio Gram-negative, Reduce sulfur; can be used for removal of

commashaped toxic and
Rod radioactive waste

Myxobacterium Gram-negative, coccoid Live in soil; can move by gliding;

bacteria forming colonies
(swarms)

Epsilonproteobacteria

The smallest class of Proteobacteria is Epsilonproteobacteria, which are gram-negative microaerophilic bacteria (meaning

they only require small amounts of oxygen in their environment). Two clinically relevant genera of Epsilonproteobacteria

are Campylobacter and Helicobacter, both of which include human pathogens.

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Campylobacter can cause food poisoning that manifests as severe enteritis (inflammation in the small intestine). This

condition, caused by the species C. jejuni, is rather common in developed countries, usually because of eating contaminated

poultry products. Chickens often harbor C. jejuni in their gastrointestinal tract and feces, and their meat can become

contaminated during processing.

Within the genus Helicobacter, the helical, flagellated bacterium H. pylori has been identified as a beneficial member of the

stomach microbiota, but it is also the most common cause of chronic gastritis and ulcers of the stomach and duodenum. H.

pylori is linked to stomach cancer. H. pylori is somewhat unusual in its ability to survive in the highly acidic environment of

the stomach. It produces urease and other enzymes that modify its environment to make it less acidic.

Genus morphology Unique characteristics

Campylobacter Gram-negative, Aerobic (microaerophilic); often infects

spiral-shaped chickens; may infect humans via
rod undercooked meat, causing severe enteritis

Helicobacter Gram-negative, Aerobic (microaerophilic) bacterium; can

spiral-shaped damage the inner lining of the
rod Gram-negative,

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 spiral-shaped stomach, causing chronic gastritis, peptic
rod
ulcers, and stomach cancer

Phototrophic bacteria are a large and diverse category of bacteria that do not represent a taxon but, rather, a group of

bacteria that use sunlight as their primary source of energy. This group contains both Proteobacteria and Nonproteobacteria.

They use solar energy to synthesize ATP through photosynthesis. When they produce oxygen, The sulfur bacteria perform

anoxygenic photosynthesis, using sulfites as electron donors and releasing free elemental sulfur. Nonsulfur bacteria use

organic substrates, such as succinate and malate, as donors of electrons.

The purple sulfur bacteria oxidize hydrogen sulfide into elemental sulfur and sulfuric acid and get their purple color from

the pigments bacteriochlorophylls and carotenoids. Bacteria of the genus Chromatium are purple sulfur

Gammaproteobacteria. These microorganisms are strict anaerobes and live in water. They use carbon dioxide as their only

source of carbon, but their survival and growth are possible only in the presence of sulfites, which they use as

electron donors.

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The green sulfur bacteria use sulfide for oxidation and produce large amounts of green bacteriochlorophyll. The genus

Chlorobium is a green sulfur bacterium that is implicated in climate change because it produces methane, a greenhouse gas.

These bacteria use at least four types of chlorophyll for photosynthesis. The most prevalent of these, bacteriochlorophyll, is

stored in special vesicle-like organelles called chlorosomes.

Purple nonsulfur bacteria are similar to purple sulfur bacteria, except that they use hydrogen rather than hydrogen sulfide for

oxidation. Among the purple nonsulfur bacteria is the genus Rhodospirillum. These microorganisms are facultative

anaerobes, which are actually pink rather than purple, and can metabolize (“fix”) nitrogen. They may be valuable in the

field of biotechnology because of their potential ability to produce biological plastic and hydrogen fuel.

The green nonsulfur bacteria are similar to green sulfur bacteria but they use substrates other than sulfides for oxidation.

Chloroflexus is an example of a green nonsulfur bacterium. It often has an orange color when it grows in the dark, but it

becomes green when it grows in sunlight. It stores bacteriochlorophyll in chlorosomes, similar to Chlorobium, and performs

anoxygenic photosynthesis, using organic sulfites (low concentrations) or molecular hydrogen as electron donors, so it can

survive in the dark if oxygen is available. Chloroflexus does not have flagella but can glide, like Cytophaga. It grows at a

wide range of temperatures, from 35 °C to 70 °C, thus can be thermophilic.

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Cyanobacteria; they get their bluegreen color from the chlorophyll contained in their cells. Cyanobacteria have other

remarkable properties. Amazingly adaptable, they thrive in many habitats, including marine and freshwater environments,

soil, and even rocks. They can live at a wide range of temperatures, even in the extreme temperatures of the Antarctic. They

can live as unicellular organisms or in colonies, and they can be filamentous, forming sheaths or biofilms. Many of them fix

nitrogen, converting molecular nitrogen into nitrites and nitrates that other bacteria, plants, and animals can use. The

reactions of nitrogen fixation occur in specialized cells called heterocysts.

Photosynthesis in Cyanobacteria is oxygenic, using the same type of chlorophyll a found in plants and algae as the primary

photosynthetic pigment. Cyanobacteria also use phycocyanin and cyanophycin, two secondary photosynthetic pigments that

give them their characteristic blue color. They are located in special organelles called phycobilisomes and in folds of the

cellular membrane called thylakoids,

Unfortunately, cyanobacteria can sometimes have a negative impact on human health. Genera such as Microcystis can form

harmful cyanobacterial blooms, forming dense mats on bodies of water and producing large quantities of toxins that can

harm wildlife and humans. These toxins have been implicated in tumors of the liver and diseases of the nervous system in

animals and humans.

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GRAM POSITIVE BACTERIA

Prokaryotes are identified as gram-positive if they have a multiple layer matrix of peptidoglycan forming the cell wall.

Crystal violet, the primary stain of the Gram stain procedure, is readily retained and stabilized within this matrix, causing

gram- positive prokaryotes to appear purple under a bright field microscope after Gram staining. Gram stain was one of the

main criteria used to classify prokaryotes, even though some prokaryotes did not readily stain with either the primary or

secondary stains used in the Gram stain procedure.

Advances in nucleic acid biochemistry have revealed additional characteristics that can be used to classify gram positive

prokaryotes, namely the guanine to cytosine ratios (G+C) in DNA and the composition of 16S Rrna subunits.

Microbiologists currently recognize two distinct groups of gram-positive, or weakly staining gram-positive, prokaryotes. The

class Actinobacteria comprises the high G+C gram-positive bacteria, which have more than 50% guanine and cytosine

nucleotides in their DNA. The class Bacilli comprises low G+C gram-positive bacteria, which have less than 50% of

guanine and cytosine nucleotides in their DNA.

Actinobacteria: High G+C Gram-Positive Bacteria

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The name Actinobacteria comes from the Greek words for rays and small rod, but Actinobacteria are very diverse. Their

microscopic appearance can range from thin filamentous branching rods to coccobacilli. Some Actinobacteria are very large

and complex, whereas others are among the smallest independently living organisms. Most Actinobacteria live in the soil,

but some are aquatic. The vast majority are aerobic. One distinctive feature of this group is the presence of several different

peptidoglycans in the cell wall.

The genus Actinomyces is a much studied representative of Actinobacteria. Actinomyces spp. play an important role in soil

ecology, and some species are human pathogens. A number of Actinomyces spp. inhabit the human mouth and are

opportunistic pathogens, causing infectious diseases like periodontitis (inflammation of the gums) and oral abscesses. The

species A. israelii is an anaerobe notorious for causing endocarditis (inflammation of the inner lining of the heart)

Genus Morphology Unique characteristics

Bifidobacterium Gram-positive, Anaerobes commonly found in human gut microbiota

filamentous
actinobacterium

Corynebacterium Gram-positive Aerobes or facultative anaerobes; form palisades; grow

bacillus slowly;
require enriched media in culture; C. diphtheriae causes

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 diphtheria

Mycobacterium Gram-positive, acidfast Slow growing, aerobic, resistant to drying and phagocytosis;
Bacillus covered with a waxy coat made of mycolic acid; M.
tuberculosis
causes tuberculosis; M. leprae causes leprosy

Nocardia Weakly gram-positive May colonize the human gingiva; may cause severe
bacillus; forms acidfast pneumonia
branches and inflammation of the skin

Micrococcus Gram-positive Ubiquitous in the environment and on the human skin;

coccus, form oxidasepositive
microscopic clusters (as opposed to morphologically similar S. aureus); some
are opportunistic pathogens

Low G+C Gram-positive Bacteria

The low G+C gram-positive bacteria have less than 50% guanine and cytosine in their DNA, and this group of

bacteria include a number of genera of bacteria that are pathogenic.

Clostridium spp. produce more kinds of protein toxins than any other bacterial genus, and several species are human

pathogens. C. perfringens is the third most common cause of food poisoning in the United States and is the causative agent

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of an even more serious disease called gas gangrene. Gas gangrene occurs when C. perfringens endospores enter a wound

and germinate, becoming viable bacterial cells and producing a toxin that can cause the necrosis (death) of tissue. C. tetani,

which causes tetanus, produces a neurotoxin that is able to enter neurons, travel to regions of the central nervous system

where it blocks the inhibition of nerve impulses involved in muscle contractions, and cause a life-threatening spastic

paralysis. C. botulinum produces botulinum neurotoxin, the most lethal biological toxin known. Botulinum toxin is

responsible for rare but frequently fatal cases of botulism. The toxin blocks the release of acetylcholine in neuromuscular

junctions, causing flaccid paralysis.

Genus Morphology Unique characteristics

Bacillus Large, gram-positive Aerobes or facultative anaerobes; form endospores; B.

anthracis causes anthrax in cattle and humans, B. cereus
bacillus may cause food poisoning

Clostridium Gram-positive bacillus Strict anaerobes; form endospores; all known species
are pathogenic, causing tetanus, gas gangrene, botulism,
and colitis

Staphylococcus Gram-positive coccus; Tolerate high salt concentration; facultative anaerobes;

forms produce catalase; S. aureus can also produce coagulase
microscopic clusters in and toxins responsible for local (skin) and generalized

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 culture that resemble infections
bunches of grapes

Streptococcus Gram-positive coccus; Diverse genus; classified into groups based on sharing
formschains or pairs in certain antigens; some species cause hemolysis and may
culture produce toxins responsible for human local (throat) and
generalized disease

Lactobacillus Gram-positive bacillus Facultative anaerobes; ferment sugars into lactic acid; part
of the vaginal microbiota; used as probiotics

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Bacterial identification

A prokaryotic species is defined simply as a population of cells with similar characteristics. The members of bacteria

species are essentially indistinguishable from each other but are distinguishable from members of other species usually on

the basis of several features. In some cases pure culture of the same species are not identical in all ways. Each such group is

called a strain which is a collection of cells derived from a single cell. Strains are identified by numbers, letter or names that

follow each other.

Bergy’s manual of determinative bacteriology provides a reference for identifying bacteria in the laboratory as well as

classification scheme for bacteria. Bergy’s manual does not classify bacteria according to evolutionary relatedness but

provides identification schemes based on such criteria as cell wall composition, morphology, differential staining, oxygen

requirement and biochemical testing.

The complete identification of bacteria involves the following steps:

(a) morphology of bacterial colony on solid medium and microscopy

(b) growth in liquid medium,

(c) biochemical reactions

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(d) molecular methods.

Morphology of Bacterial Colony on Solid Medium

When classifying microorganisms, all known characteristics are taken into consideration, but certain differential and

distinguishable characteristics are selected and used for the purpose of identification. Primary identification usually involves

a few simple but fundamental tests such as cellular morphology (usually shown by Gram (bacteria) growth under various

atmospheric conditions, growth on various types of culture media (for example MacConkey agar, Sabouraud agar plate

culture), catalase and oxidase

Microscopic study and staining reveals the shape (coccus or rod) and the characteristic grouping and arrangement of the

cells, their size and the presence of intracellular inclusions, for example spores. In addition to morphology, the Gram stained

preparation also divides bacteria in two categories - the Gram positive and the Gram negative bacteria1

There are a number of staining methods used to identify bacteria

Differential staining
These are stains that react differentially with different kinds of bacteria in order to distinguish among them

Gram stain- Procedure

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Add crystal violet stain over the fixed smear on a slide. Let stand for 60 seconds; Pour off the stain and gently rinse the

excess stain with a stream of water. Add the iodine solution on the smear, enough to cover the fixed culture. Let stand for 60

seconds. Pour off the iodine solution and rinse the slide with running water. Shake off the excess water from the

surface.Add a few drops of decolorizer 95% absolute ethanol so the solution trickles down the slide. Rinse it off with water

after 5 seconds. Counterstain with safranin solution for 40 seconds. Wash off the solution with water. Blot with filter paper

to remove the excess water. Observe under microscope.

Gram positive will retain the crystal violet stain and appear purple while the gram negative appear pink because they were

unable to retain the crystal violet stain but took up the safranin.

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 Acid fast

2. Ziehl–Neelsen stain
Used to differentiate between acid fast bacilli and non acid fast bacilli. Used to distinguish mycobacterium species and
some species of norcadia. The experiment involves the bacteria been treated with carbol fuchsin and with acid alcohol. Acid
fast bacteria will remain red because they retain the carbol fuchsin colour while the non acid fast will be blue which is the
colour of methylene blue.

Mycobacterium tuberculosis is an example of acid fast bacteria.

Procedure
Add one loopful of sterile water to a microscope slide. 2. Make a heavy smear of Mycobacterium smegmatis. Mix thoroughly with your loop.
Then transfer a small amount of Staphylococcus epidermidis to the same drop of water. You will now have a mixture of M. smegmatis and S.
epidermidis. 3. Air dry and heat fix well. 4. Cover the smear with carbolfuchsin dye. Place a piece of paper towel on top of the dye. Be sure the
paper towel is saturated with the dye. Carbolfuchsin is a potential carcinogen. Please wear gloves when working with this dye. 5. Place the slide

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on the rack over dry heat for 2 minutes. 6. Cool and rinse with water. 7. Decolorize by placing a drop of acid alcohol on the slide and allowing it
to sit for 15 seconds. 8. Wash the top and bottom of slide with water and clean the slide bottom well. 9. Counterstain with Methylene Blue for 30
seconds to 1 minute. 10. Wash and blot the slide with bibulous paper. 11. Focus 10X - then use oil immersion.

Special stains
These are stains used to color and isolate various structures such as capsule, endospore and flagella.
a) Negative staining- used to demonstrate capsule. Since most capsules do not accept most stains, they appear as
unstained halos around bacterial cells

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b) Endospore staining
This is a method used to detect the presence of endospores. When malachite green is applied to a heat fixed smear of bacterial cells. The stain
penetrates the endospore and stain them green.

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85
Flagella stain
In this method a mordant is used to build up the diameter of flagella until they become visible microscopically when
stain with carbolfuuchsin
Alternatively a wet mount method can be used.

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Morphological characteristics such as colony morphology

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Morphological characteristics such as shape and size

The size of bacterial colonies, assuming favourable growth conditions, is generally uniform within a species. For

example Streptococcus species are small, usually 1mm in diameter, whilst Staphylococcus species and species within

the family, Enterobacteriaceae are larger and usually 2 - 3mm in diameter, and those of Bacillus species are much

larger in size and usually 2 - 7mm in diameter.

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Microorganisms can be grouped on the basis of their growth requirements.
It is usual to divide organisms into five categories according to their atmospheric requirements:
• strict aerobes grow only in the presence of oxygen
• strict anaerobes grow only in the absence of oxygen
• facultative organisms grow aerobically or anaerobically
• microaerophilic organisms grow best in an atmosphere with reduced oxygen concentration (addition of 5-10% CO2 may
enhance growth)

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• carboxyphilic (or capnophilic) organisms require additional CO2 for growth

Temperature1
Organisms may also be divided according to their temperature requirements:
• psychrophilic organisms grow at low temperatures 2-5°C (optimum 10-30°C).
• mesophilic organisms grow at temperatures between 10-45°C (optimum 30-40°C).
• thermophilic organisms grow very little at 37°C (optimum 50-60°C).
• hyperthermophilic organisms grow at temperatures of 80°C or higher

Most clinically encountered organisms are mesophilic.

Motility

Many bacteria are observed to be motile and move from one position to another when suspended in fluid. True motility

must not be confused with Brownian movement (vibration caused by molecular bombardment) or convection currents.

Microscopic examination may indicate whether a motile organism has polar flagellae shown by a darting zigzag movement

or peritrichate flagellae, which cause a less vigorous and more vibratory movement. Some bacteria may be motile at

different temperatures, for example motile at ambient temperature but not at 37°C, or vice versa.
1
Nutrition

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Study of the nutritional requirements of an organism is useful in identification, for example the ability to grow on ordinary

nutrient media, the effect of adding blood,serum or glucose or the necessity for specific growth factors .

Biochemical tests used to identify Gram Positive Bacteria

Catalase Test

This test is used to identify organisms that produce the enzyme, catalase. This enzyme detoxifies hydrogen peroxide by

breaking it down into water and oxygen gas.

The bubbles resulting from production of oxygen gas clearly indicate a catalase positive result. The sample on the right

below is catalase positive. The Staphylococcus spp. and the Micrococcus spp. are catalase

positive. The Streptococcus and Enterococcus spp. are catalase negative.

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Mannitol Salt Agar (MSA)

This type of medium is both selective and differential. The MSA will select for organisms such as Staphylococcus species which
can live in areas of high salt concentration (plate on the left in the picture below). This is in contrast to Streptococcus species,
whose growth is selected against by this high salt agar (plate on the right in the picture below).

The differential ingredient in MSA is the sugar mannitol. Organisms capable of using mannitol as a food source will produce acidic
byproducts of fermentation that will lower the pH of the media. The acidity of the media will cause the pH indicator, phenol red, to
turn yellow. Staphylococcus aureus is capable of fermenting mannitol (left side of left plate) while Staphylococcus epidermidis is not
(right side of left plate).

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Nitrate reduction test

It utilizes nitrate broth. This is a differential medium. It is used to determine if an organism is capable of reducing nitrate

(NO3-) to nitrite (NO2-) or other nitrogenous compounds via the action of the enzyme nitratase (also called nitrate reductase).

This test is important in the identification of both Gram-positive and Gram-negative species.

After incubation, these tubes are first inspected for the presence of gas in the Durham tube. In the case of nonfermenters,

this is indicative of reduction of nitrate to nitrogen gas. However, in many cases gas is produced by fermentation and

93
further

94
testing is necessary to determine if reduction of nitrate has occurred. This further testing includes the addition of sulfanilic

acid (often called nitrate I) and dimethyl-alpha-napthalamine (nitrate II). If nitrite is present in the media, then it will react

with nitrate I and nitrate II to form a red compound. This is considered a positive result. If no red color forms upon addition

of nitrate I and II, this indicates that either the NO 3- has not been converted to NO2- (a negative result), or that NO3- was

converted to NO2- and then immediately reduced to some other, undetectable form of nitrogen (also a positive result). In

order to determine which of the preceding is the case, elemental zinc is added to the broth. Zinc will convert any remaining

NO3- to NO2- thus allowing nitrate I and nitrate II to react with the NO2- and form the red pigment (a verified negative

result). If no color change occurs upon addition of zinc then this means that the NO3- was converted to NO2- and then was

converted to some other undetectable form of nitrogen (a positive result).

If the nitrate broth turns red (tubes pictured in the center) after nitrate I and nitrate II are added, this color indicates a

positive result. If instead, the tube turns red (tube pictured on the left) after the addition of Zn, this indicates a negative

result. If there is no color change in the tube after the addition of nitrate I and nitrate II, the result is uncertain. If the tube is

colorless (picture on the right) after the addition of Zn this indicates a positive test

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Starch hydrolysis test

This test is used to identify bacteria that can hydrolyze starch (amylose and amylopectin) using the enzymes -amylase and oligo-
1,6-glucosidase. Often used to differentiate species from the genera Clostridium and Bacillus. Because of the large size of amylose
and amylopectin molecules, these organisms can not pass through the bacterial cell wall. In order to use these starches as a
carbon source, bacteria must secrete-amylase and oligo-1,6-glucosidase into the extracellular space. These enzymes break the
starch molecules into smaller glucose subunits which can then enter directly into the glycolytic pathway. In order to interpret the
results of the starch hydrolysis test, iodine must be added to the agar. The iodine reacts with the starch to form a dark brown color.
Thus, hydrolysis of the starch will create a clear zone around the bacterial growth. Bacillus subtilis is positive for starch hydrolysis
(pictured below on the left). The organism shown on the right is negative for starch hydrolysis.

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Motility agar

This a differential medium used to determine whether an organism is equipped with flagella and thus capable of swimming

away from a stab mark. The results of motility agar are often difficult to interpret. Generally, if the entire tube is turbid, this

indicates that the bacteria have moved away from the stab mark (are motile). The organisms in the two tubes pictured on the

right are motile. If, however, the stab mark is clearly visible and the rest of the tube is not turbid, the organism is likely

nonmotile (tube pictured on the left).

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Coagulase test

Coagulase is an enzyme that clots blood plasma. This test is performed on Gram-positive, catalase positive species to

identify the coagulase positive Staphylococcus aureus. Coagulase is a virulence factor of S. aureus. The formation of clot

around an infection caused by this bacteria likely protects it from phagocytosis. This test differentiates Staphylococcus

aureus from other coagulase negative Staphylococcus species.

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Biochemical tests to identify Gram negative bacteria

Oxidase Test

This test is used to identify microorganisms containing the enzyme cytochrome oxidase (important in the electron transport
chain). It is commonly used to distinguish between oxidase negative Enterobacteriaceae and oxidase
positive Pseudomadaceae.

Cytochrome oxidase transfers electrons from the electron transport chain to oxygen (the final electron acceptor) and reduces
it to water. In the oxidase test, artificial electron donors and acceptors are provided. When the electron donor is oxidized by
cytochrome oxidase it turns a dark purple. This is considered a positive result. In the picture below the organism on the right
(Pseudomonas aeruginosa) is oxidase positive.

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Glucose broth with Durham tubes

This is a differential medium. It tests an organism's ability to ferment the sugar glucose as well as its ability to convert the

end product of glycolysis, pyruvic acid into gaseous byproducts. This is a test commonly used when trying to identify

Gram- negative enteric bacteria, all of which are glucose fermenters but only some of which produce gas.

10
Like MSA, this medium also contains the pH indicator, phenol red. If an organism is capable of fermenting the sugar

glucose, then acidic byproducts are formed and the pH indicator turns yellow. Escherichia coli is capable of fermenting

glucose as are Proteus mirabilis (far right) and Shigella dysenteriae (far left). Pseudomonas aeruginosa (center) is a

nonfermenter.

The end product of glycolysis is pyruvate. Organisms that are capable of converting pyruvate to formic acid and formic acid

to H2 (g) and CO2 (g), via the action of the enzyme formic hydrogen lyase, emit gas. This gas is trapped in the Durham tube

and appears as a bubble at the top of the tube. Escherichia coli and Proteus mirabilis (far right) are both gas producers.

Notice that Shigella dysenteriae (far left) ferments glucose but does not produce gas.

*Note - broth tubes can be made containing sugars other than glucose (e.g. lactose and mannitol). Because the same pH

indicator (phenol red) is also used in these fermentation tubes, the same results are considered positive (e.g. a lactose broth

tube that turns yellow after incubation has been inoculated with an organism that can ferment lactose).

10
MacConkey agar

This medium is both selective and differential. The selective ingredients are the bile salts and the dye, crystal violet which

inhibit the growth of Gram-positive bacteria. The differential ingredient is lactose. Fermentation of this sugar results in an

acidic pH and causes the pH indicator, neutral red, to turn a bright pinky-red color. Thus organisms capable of lactose

10
fermentation such as Escherichia coli, form bright pinky-red colonies (plate pictured on the left here). MacConkey agar is

commonly used to differentiate between the Enterobacteriaceae.

Organism on left is positive for lactose fermentation and that on the right is negative.

Nitrate broth

See as above

Motility

See above

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Simmon’s Citrate Agar

This is a defined medium used to determine if an organism can use citrate as its sole carbon source. It is often used to

differentiate between members of Enterobacteriaceae. In organisms capable of utilizing citrate as a carbon source, the

enzyme citrase hydrolyzes citrate into oxaoloacetic acid and acetic acid. The oxaloacetic acid is then hydrolyzed into

pyruvic acid and CO2. If CO2 is produced, it reacts with components of the medium to produce an alkaline compound (e.g.

Na2CO3). The alkaline pH turns the pH indicator (bromthymol blue) from green to blue. This is a positive result (the tube on

the right is citrate positive). Klebsiella pneumoniae and Proteus mirabilis are examples of citrate positive organisms.

Escherichia coli and Shigella dysenteriae are citrate negative.

10
Urease test

This test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease. It is commonly used to

distinguish the genus Proteus from other enteric bacteria. The hydrolysis of urea forms the weak base, ammonia, as one of

its products. This weak base raises the pH of the media above 8.4 and the pH indicator, phenol red, turns from yellow to

pink. Proteus mirabilis is a rapid hydrolyzer of urea (center tube pictured here). The tube on the far right was inoculated

with a urease negative organism and the tube on the far left was uninoculated.

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Sulfur Indole Motility Media (SIM)

This is a differential medium. It tests the ability of an organism to do several things: reduce sulfur, produce indole and swim
through the agar (be motile). SIM is commonly used to differentiate members of Enterobacteriaceae.

Sulfur can be reduced to H2S (hydrogen sulfide) either by catabolism of the amino acid cysteine by the enzyme cysteine
desulfurase or by reduction of thiosulfate in anaerobic respiration. If hydrogen sulfide is produced, a black color forms in
the medium. Proteus mirabilis is positive for H2S production. The organism pictured on the far left is positive for hydrogen
sulfide production.

Bacteria that have the enzyme tryptophanase, can convert the amino acid, tryptophane to indole. Indole reacts with added
Kovac’s reagent to form rosindole dye which is red in color (indole +). Escherichia coli is indole positive. The organism
pictured second from left is E. coli and is indole positive.

SIM tubes are inoculated with a single stab to the bottom of the tube. If an organism is motile than the growth will radiate
from the stab mark and make the entire tube appear turbid. Pseudomonas aeruginosa and the strain of Proteus mirabilis that
we work with are motile.

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Molecular techniques

16S ribosomal RNA (16S rDNA) sequencing

16S rDNA gene sequencing is a genotypic identification method and the hyper variable regions of 16S rDNA gene

sequences provide species-specific signature sequences useful for bacterial identification It has been used for identifying

bacteria or for phylogenetic studies and has subsequently been found to be capable of re-classifying bacteria into completely

new species, or even genera. For bacterial identification, 16S rDNA sequencing is particularly important in the case of

bacteria with unusual phenotypic profiles, rare bacteria, slow-growing bacteria, as well as culture-negative infections. It has

also been used to describe new bacteria that have never been successfully cultured in laboratories.

Despite its applicability, sequencing 16S ribosomal DNA lacks widespread use due to the technical and cost issues. Other

drawbacks are difficulty in interpretation of results for most technicians and clinical microbiologists and lack of automation

of the technology so that it could be used routinely in clinical microbiology laboratories27. Its usefulness is also limited

because of the high percentage of sequence similarity between some closely related species.

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