SCHOOL OF HEALTH SCIENCES

GTB110/3
BASIC MICROBIOLOGY

PRACTICAL (1): PART C

BACTERIOLOGY

ASSOC. PROF. DR. NURUL ASMA ABDULLAH

INTRODUCTION

I. Microscopic methods of examining bacteria

The human eye cannot see bacteria without the use of magnification.
Microscopes are used to see bacteria whose average size is 0.5 to 2.0 µm and
whose average diameter ranges from 0.3 to 1.0 µm. Microscopes are used in
many research and clinical laboratory to study and viewing stained and unstained
bacteria, observing bacterial morphology and cell arrangement, counting and
measuring bacterial cells. Because bacteria have a refractive index similar to that
of water, biological stains are often needed so that the bacteria can be
visualized. Stains are organic or aqueous preparations of dyes that impart a
variety of colours to the bacterial cells and to make some structures more visible.
A bacterial smear (is a thin film material containing the microorganisms spread
over the surface of the slide) is prepared and fixed (attached) to the microscopic
slide before the bacteria can be stained. Fixing kills the bacteria and attached
them to the slide. Stains are either basic dyes (positive ions) or acidic dyes
(negative ions). The colored positive ions in basic dyes attracted to the negatively
charged bacterial cells. Basic dyes include: crystal violet, methylene blue,
malachite green, and safranin. Acidic dyes are eosin acid fuchsin, and nigrosin.
Negative staining is produced when the colored negative ion of an acidic dye will
stain the background of the bacterial smear.

Microbiologists use three kinds of staining techniques; simple, differential, and

special stains:

Simple stains

A simple stain is an aqueous or alcohol solution of a single basic dye. It is used

to visualize cellular shapes and arrangements. A mordant is used to increase the
affinity of a stain for a biological specimen. Common simple stains used are
methylene blue, carbol fuchsin, crystal violet, and safranin.

Differential stains

Differential stains such as Gram-stain and acid-fast stain (Ziehl- Neelson),

differentiate bacteria according to their reactions to the stains. The Gram-stain
procedure uses a purple stain (crystal violet), iodine as a mordant, an alcohol
decolorizer and counterstain (safranin). Gram-positive bacteria retain the purple
stain after the decolorization step; Gram-negative bacteria do not and thus
appear pink from the counterstain.
Acid-fast microbes such as members of the genera Mycobacterium and
Nocardia, retain carbol fuchsin after acid-alkohol decolorization and appear red;
non-acid-fast microbes take up the methylene blue counterstain and appear blue.
Making a smear

1. Clean a plain microscope slide thoroughly using lens tissue.

2. Label a microscope slide with a marker pen to record the culture being used,
date and initials; this is also a useful reminder of which side of the slide is being
used.
3. Flame a wire loop to ensure that no culture accidentally remains from a
previous operation.
4. Transfer one or two loopfuls of tap water on to the centre of the slide.
5. Flame loop and allow to cool.

6. Using aseptic technique, transfer a very small part of a single colony from a
plate or slope of agar medium into the tap water. If the amount of culture on the
loop is easily visible you have taken too much!
7. Make a suspension of the culture in the tap water on the slide and thoroughly
but gently spread it evenly over an oval area of up to 2 cm length.
8. Flame the loop. If it is necessary to use a liquid culture or sample, the use of
tap water to prepare the smear will probably be unnecessary and may result in a
smear with too few cells.
9. Dry the suspension by warming gently over a Bunsen burner flame and then
“fix” it by quickly passing it through the flame a few times. This is called a heat-
fixed smear; it should be visible to the naked eye as a whitish area. Fixing is
necessary to ensure that cells adhere to the slide and to minimize any post
mortem changes before staining. The smear is now ready to be stained

HINTS:
Always use a young culture because older cultures of Gram-positive bacteria
tend to lose the ability to retain the crystal violet-iodine complex and appear to be
Gram-negative; but some bacteria are naturally only weakly Gram-positive. The
amount of alcohol treatment (the differential stage) must be judged carefully
because over-treatment washes the crystal violet-iodine complex from Gram-
positive bacteria and they will appear to be Gram-negative.

Take care to make an even smear otherwise alcohol will continue to wash the
violet/purple colour from thick parts of the smear while thin parts are being over-
decolorised. At the end of the procedure, check that the labeling has not been
washed off by the alcohol.
Don’t despair if the stained smear is not visible to the naked eye; this may
happen with a Gram-negative reaction.

Staining specific structures

Some staining techniques are specific for particular structures like bacterial
capsules, flagella and endospores. The negative staining technique is used to
reveals the presence of the capsule staining, using India ink or nigrosin dye.
Spore staining methods use the Schaeffer-Fulton procedure by heating the
bacteria with malachite green and safranin counterstain. This technique yields a
green endospore with pink or red cell. Flagella staining procedures provide
taxonomically valuable information about the presence and distribution pattern of
flagella

II. Culture Media

In the laboratory, the nutrient preparations that are used for culturing are called
media (singular, medium). Three physical forms are used: liquid, semi-solid, and
solid media. Microorganisms may be cultured using two different types of media.
Chemically defined media are composed of known amounts of pure chemicals. In
routine bacteriology laboratory exercises, complex media are employed and are
composed of complex materials that are rich in nutrients and vitamins. For the
isolation and identification of a particular microorganism, especially those from
patients with infectious diseases or samples from food and water, and
environmental studies, selective, differential, or enrichment media are often used.
Such special media are an essential part of modern diagnostic media:

Supportive media (general isolation media)

This media support the growth of most non-fastidious bacteria. Examples include
nutrient agar and nutrient broth. No advantage is given to any group of bacteria.

Enrichment media (non-selective isolation media)

This media contain a nutrient supplement. Examples include sheep blood agar
(SBA) and chocolate agar (heated blood). This media also support the growth of
fastidious bacteria. Selenite broth and thioglycollate broth are some examples of
an enrichment broth.

Differential media

This media provide distinct colonial appearances of bacteria to aid in their

identification. Most differential media are used to isolate Gram negative bacteria
through the addition of (a) compound(s) that is (are) inhibitory for Gram positive
bacteria such as MacConkey agar (Mac), Eosin-Methylene Blue agar (EMB), and
inhibition of normal flora such as Salmonella-Shigella (SS) agar and Xylose-
Lysine-Deoxycholate (XLD) agar.
Bacterial growth can be observed as discrete colonies on agar media, which is
essential to obtain isolated colonies or to enumerate colonies. Colonial
characteristics are important observation in the identification of bactera. It is
essential to make observation under direct light, preferably with a hand lens or a
dissecting microscope. Some important descriptions include the form, elevation,
margin, surface, consistency, density and colour.
III. Biochemical tests

A pure bacterial culture growing on a medium can be used to inoculate a series

of biochemical tests to gather information about the bacteria’s phenotypic
characteristics. By inoculating these biochemical tests, microbiologist can learn
what enzymes the organism does or does not possess. A bacteria ability to
produce or not produce a particular enzyme is part of that bacteria phenotype.
Most metabolic reactions are catalyzed by enzymes and this will determine which
substrates the bacteria can catabolized. The metabolism or use of these organic
molecules often produces by-products that can be very helpful in identification
and characterization of bacteria. Examples include oxidase, catalase, urease,

IV. Serology tests

Serology tests help to diagnose infectious diseases (eg; cause by bacteria) by

detecting either antigens or antibodies in clinical specimens. The test performed
on serum specimens are sometimes called serologic procedure. Some serology
tests are designed to detect antigens, whereas others detect antibodies.
Detection of a particular pathogen’s antigens in a clinical specimen is an
indication that the pathogen is present in the patient, thus providing direct
evidence that the patient is infected with that pathogen. Detection of antibodies
directed against a particular pathogen is indirect evidence of infection with that
pathogen. Three possible explanations for the presence of antibodies to a
particular pathogen are present infection, past infection and vaccination. A
variety of different serology tests have been designed so that visible reaction will
be observed if an antigen-antibody reaction takes place. They include
agglutination, precipitin, immunodiffusion, immunoelectrophoresis,
immunofluorescence procedure and ELISA.

OBJECTIVES

1. Appreciate the bacteria morphological diversity through the use of microscopy

and various staining techniques.

2. Describe the different types of culture media, their composition and use.

3. Appreciate the diversity in bacterial metabolic pathways based on biochemistry

tests provided.

4. Appreciate basic serology tests displayed during the laboratory session.

LABORATORY REPORT
Students are required to prepare a short laboratory report from all their
observations and what they have learned from each section of the laboratory.
Laboratory report is to be submitted within two weeks from the practical date.

I. Microscopy and Staining

1A. Making smear and Gram Staining

Perform the Gram staining procedure for organisms listed below and draw the
Gram-stained bacteria in the following boxes and describe the magnification,
Gram type, and cell morphology and arrangement.

a. Staphylococcus aureus Magnification:

Gram type:
Cell shape:
Cell arrangement:

b. Escherichia coli Magnification:

Gram type:
Cell shape:
Cell arrangement:

1B. Observe the prepared slides of bacteria under microscope and draw the
Gram stained bacteria in the following boxes and describe the magnification,
Gram type and cell morphology and arrangement.

a. Streptococcus pyogens Magnification:

Gram type:
Cell shape:
Cell arrangement:

Magnification:
b. Micrococcus sp Gram type:
Cell shape:
Cell arrangement:

Magnification:
c. Neisseria sp Gram type:
Cell shape:
Cell arrangement:

d. Pseudomonas species Magnification:

Gram type:
 Cell shape:
Cell arrangement:

1C. Acid Fast Bacilli Staining (Ziehl Neelson)- for Mycobacterium

tuberculosis and FLM of special staining (Endospore and flagella staining)
- Perform AFB staining procedure and report the observation.
- Read and understand the special staining methods given.

Mycobacterium tuberculosis Magnification:

Gram type:
Cell shape:
Cell arrangement:

Endospore staining

Flagella staining

II. Culture Media

Record your observation of the growth of the bacteria on each media. Define the
type of culture media displayed during the laboratory session and describe the
principal of each media. Also explain the importance of differentiating the
bacteria on each media.

Media Observation (eg; photograph)

MacConkey Agar E. coli

Salmonella
Definition: Klebsiella
Principal:
Importance:

Thiosulphite Citrate Bile Sucrose Vibrio cholera

(TCBS) Vibrio parahemolyticus

Definition:
Principal:
Importance:
Hektoen Enteric (HE) Salmonella typii
Salmonella spp
Definition:
Principal:
Importance:

Blood Agar Staphylococcus

Streptococcus pyogenes
Definition: Strep. pneumoniae
Principal:
Importance:

Martin Thayer Agar Neisseria gonorrhoeae

Definition:
Principal:
Importance:

Chocolate Agar Haemophilus influenza

H. parainfluenzae
Definition:
Principal:
Importance:

*(Note: You are also required to find additional information on bacteria not
provided during the laboratory session)

III. Biochemical Tests

Record your observation and complete the following table. Describe the principal,
purpose and interpretation of each test.

Table1: TSI test

Bacterium Carbohydrate Fermentation H2S Production

Butt Colour Slant Colour Black H2S
E. coli
Klebsiella sp
S. typhi

Table 2: Citrate test

Bacterium Presence/Absence of Growth Colour of Citrate Use

medium (+) or (-)
E. coli
Klebsiella sp
Table 3: SIM test

Bacterium Citrate Use Indole Motility

(+) or (-) Colour of Indole (+) or (-) Motile Non-Motile
Reagent Layer
E. coli
Klebsiella sp
Proteus sp

Table 4: MR / VP test

Bacterium Methyl Red Test Voges-Proskauer Test

Medium Colour + or - Medium Colour + or -
E. coli
Klebsiella sp

Table 5: Urease test

Bacterium Colour of medium Urease (+) or (-)

E. coli
Klebsiella sp

IV. Serology

Explain the principle and purpose of the serology tests below.

I. Anti - Streptolysisn O Titration test (ASOT):

i. Principal
ii. Purpose
iii. Interpretation
iv. Bacteria used as + and – control

II. Rapid Plasma Reagent (RPR)

i. Principal
ii. Purpose
iii. Interpretation
v. Bacteria used as + and – control

III. Typhi Dot

i. Principal
 ii. Purpose
iii. Interpretation
iv. Bacteria used as + and – control

QUESTIONS:

1. What are the two purpose of heat fixation?

i.

ii.

2. What is the purpose of simple staining?

3. Why are basic dyes more successful in staining bacteria than acidic dyes?

4. What is the difference between a simple and differential stain?

5. Name the reagent used and state the purpose of each of the following in the
Gram stain:

a. mordant

b. primary stain

c. decolorizer

d. counterstain

6. What part of a bacterial cell is most involved with Gram staining, and why?
7. Why must young cultures be used when doing a Gram stain?

8. What is the purpose of heat during the acid-fast staining procedure?

9. What is the function of the counterstain in the acid-fast staining procedure?

10. Are acid-fast bacteria gram positive or gram negative? Explain your answer.

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