Microbial physiology/practice
Lec (2)
Acid Fast Stain
Many bacterial cells are easily stained with simple stains or using the Gram stain. A
few types of bacteria, such as the mycobacteria and Nocardia species, do not stain
using these techniques or, if stained, they produce a variable reaction because their
walls are not permeable to dyes therfor must used stain for this bacteria called Acid
fast stain or Ziehl-Neelsen Stain.
Principle of Acid Fast Stain
Acid-fast mycobacteria contain mycolic acid in their outer membrane, making the
cells waxy and resistant to staining with aqueous based stains such as the Gram stain.
The primary stain, carbolfuchsin is applied to the cells, and heat are used to allow the
stain to penetrate into the waxy surface of acid-fast microorganisms. The excess stain is
removed with treatment by acid alcohol (ethanol and hydrochloric acid). A secondary
stain, methylene blue, is then applied to the cells.
Procedure
1. Prepare and fix the specimen smear to staining.
2. Place a small strip of filter paper over the top of the specimen, and put the slide over a
boiling hot water bath on a mesh surface.
3. Cover the filter paper with the primary stain, carbolfuchsin. Leave the slide on the
water bath for 3 to 5 minutes. Continue to apply stain if the filter paper begins to dry.
4. Remove the filter paper and rinse the slide with water until the solution runs clear.
5. Put acid-alcohol decolorize over the slide for approximately 10 to 15 seconds.
6. Rinse the slide with water then Cover the smear with the secondary or counterstain,
methylene blue, for 1 minute.
7. Gently rinse the slide with water then Blot the slide dry with bibulous paper.
Result Interpretation of Acid Fast Stain
Acid fast: Bright red to intensive purple, Red, straight or rods, singly or small groups.
Non-acid fast: Blue color; In addition, background material should stain blue.
Limitations
1. The filter paper must remain moist and in contact with the specimen during heating to
allow for proper penetration of the primary stain.
2. Organisms cultivated on blood agar may experience nutrient deprivation, resulting in a
lower lipid content in the outer membrane resulting in poor staining.
Examples Acid-fast: Mycobacterium tuberculosis
Non-Mycobacterial bacteria: Nocardia
Coccidian Parasites: Cryptosporidium
1
Microbial physiology/practice
Lec (2)
Osmolarity
Osmolarity describes the total concentration of solutes in a solution. A solution
with a low osmolarity has fewer solute particles per liter of solution, while a
solution with a high osmolarity has more solute particles per liter of solution.
When solutions of different osmolarities are separated by a membrane permeable
to water, but not to solute, water will move from the side with lower osmolarity to
the side with higher osmolarity, through pressure called osmosis is the net
movement of water across a semipermeable membrane from an area of lower
solute concentration to an area of higher solute particles concentration.
While In biology labs, it’s often helpful to think about how solutions will affect
water movement into and out of cells, The ability of an extracellular solution to
make water move into or out of a cell by osmosis is known as its tonicity also
refer to (Tonicity) the concentration of a solution as compared to another solution
Which is a different from osmolarity because it takes into account both relative
solute concentrations and the cell membrane’s permeability to those solutes.
Tonicity could effect on the bacteria through the plasma membrane ,it function
is a s a selective permeability barrier that regulates the passage of substances,
water and uncharged molecules into and out of the cell, To explain this, it can be
divided into three states: hypertonic, hypotonic, and isotonic
If a bacterial cell is placed in a hypertonic solution, there will be a net flow of
water out of the cell, and the cell will lose volume. A solution will be hypertonic
to a cell if its solute particles concentration is higher than that inside the cell, so
the cell shrinks the protoplast of the cell from its wall, so the situation is
called Plasmolysis
If a bacterial cell is placed in a hypotonic solution, there will be a net flow of
water into the cell, and the cell will gain volume. lt, there is swelling of the cell,
which leads to the burst of the cell, where the osmotic pressure is high.
2
Microbial physiology/practice
Lec (2)
If a bacterial cell is placed in an isotonic solution, there will be no net flow of
water into or out of the cell, and the cell’s volume will remain stable. If the solute
concentration outside the cell is the same as inside the cell, and the solutes cannot
cross the membrane, then that solution is isotonic to the cell.
Diagram of red blood cells in hypertonic solution (shriveled), isotonic solution (normal),
and hypotonic solution (puffed up and bursting).
Materials:
1- A 24-48 hour liquid culture of E. coli, Staph.aureus.
2- Petri dishes from the nutrients containing different concentrations of
Nacl (2.5-5-7.5-10-12.5.-15%).
3- Wax pencil, inoculating loop, Bunsen burner.
The method of work:
1- Divide each plate into two parts with a wax pen from the bottom and
write the name of the bacteria, the date of cultivation and the
concentration.
2- A part of the bacterial culture is transferred by the loop to the plates
and streaking.
3- The dishes were incubated in the incubator for a period of 24-48 hours,
at 37 ° C.
After incubation, the growth results are examined and observations are
recorded.
