Bacteriology Laboratory

[Link] Esmaeili

Gram Staining Steps

1. Prepare a smear of bacteria on a slide
2. Apply crystal violet dye to the slide for 1 minute.
3. Rinse the slide with distilled water.
4. Apply iodine solution to the slide for 1 minute
5. Rinse the slide with distilled water.
6. Decolorize the slide with 70% ethanol for 10-20 seconds (or until
no more color comes out).
7. Rinse the slide with distilled water.
8. Apply safranin dye to the slide for 1 minute.
9. Rinse the slide with distilled water.
10. After the slide dries, place it under a microscope and examine it
with a 100 oil lens.
Note: Gram-positive bacteria appear purple and gram-negative
bacteria appear red.
Preparation of Microbial Smear
1. First, draw a line on the left side of the slide with a marker
2. Place a small drop of distilled water in the middle of the slide.
Then sterilize the fildoplatin-loop. in a flame and take a very small
amount of the bacterial culture.
3. Add the collected sample to the drop of water and mix it with it.
As soon as you see the water drop darkening, stop stirring. Sterilize
the fildoplatin- needle on the flame.
4. Dry the prepared slide in the presence of air.
5. After drying, hold the slide with the help of forceps and pass it
over the flame two or three times. The purpose of heating the slide
and passing it over the flame is to ensure that the microbes stick to
the slide and stabilize it and do not come off during staining and
washing.
Capsule Staining

Nigrosin staining
1. First, place a small drop of nigrosin (5 or 10%) on a clean and dry
slide.
2. Pick up a colony of encapsulated bacteria (such as Klebsiella)
with fildoplatin-loop and dissolve it in a drop of nigrosin to prepare
a suspension.
3. Place a coverslip on it.
4. Observe the coverslip with a 40 lens.
Note: Transparent capsule seen on black background

Acid-fast staining according to Ziehl-Neelsen staining method

CMN bacteria have mycolic acid in their cell walls and are acid-fast.
These bacteria are resistant to staining and are also resistant to acid
decolorization.

Acid –fast Staining steps

1. Fix the desired sample, such as sputum, on the slide using heat.
2. Fill the smear surface with fuchsin dye and heat it gently using an
flame. In such a way that steam comes out of it but does not boil and
the color of the slide surface does not dry. Continue this for 5
minutes.
3. Wash and clean the slide surface using water. Then fill its surface
with methylene blue and leave it for 1 minute.
4. Empty the color of the slide surface, wash it well with water and
let it dry at room temperature, then pour some immersion oil on its
surface and examine it with a microscope magnification of 1000 for
the presence of red bacilli on a blue background.
Spore Staining
A spore is a very strong body that develops inside some bacterial
cells and is found throughout the Bacillaceae and Clostridiacea
families.

Writz or Malachite Green Staining

It is used to observe and stain spores in bacteria.
Steps:
1. To prepare the smear, it is best to add a small amount of sterile
distilled water to the tube containing the solid culture using a
Pasteur pipette or Loop, suspend the bacteria using loop, then
place a drop of the above suspension on the slide and dry it in
the presence of air.
2. Fix the smear with heat or pure alcohol.
3. Place a piece of filter paper on the smear and add the malachite
green solution.
In the laboratory, you will be shown how to heat the prepared fruit.
You must be careful not to boil the dye solution and dry it on the
sample. To do this, you must avoid boiling it and add dye solution to
it regularly (for 5 minutes).
4. Remove the filter paper and rinse the slide with distilled water.
5. Add the safranin solution for 30-60 seconds.
6. Rinse with distilled water and dry in the presence of air.
7. Observe with a 100 lens.
Note: The use of filter paper is to prevent the dye from depositing on
the slide and the use of heat is due to the destruction of the cell wall
and the penetration of the dye into the cell.
Comparison: In gram staining, the spore is colorless because the cell
wall is not torn and the spore is not stained. After spore staining, the
spores are observed in green (they have taken on the malachite
green color).
Pure culture
The goal of pure culture is to obtain a single colony.
Four-stage culture is a widely used method that is suitable for
obtaining a single colony and observing changes caused by bacterial
growth in the culture medium, such as hemolysis.
Pure Culture

4-stage linear cultivation:

1. Hold the metal loop over the blue part of the flame until it is
hot. Allow the loop to cool.

2. Place the tube containing the culture medium on the table and
lift the lid slightly. Use the lid as a shield against air
contamination.

3. Place the loop dipped in culture medium on the agar surface.

Starting at the edge of the plate, gently drag the loop across the
agar surface. Be careful not to cut the agar surface.
 4. Remove the loop and replace the plate lid. Heat the loop again
to sterilize it. It is important to heat the loop thoroughly
because there are many bacteria on the loop ring.

5. Rotate the plate slightly. (less than 90 degrees). Allow the loop to
cool. You can touch the loop to a part of the agar where there are no
bacteria to cool it. Start another zigzag pattern at the end of the first
pattern.
[Link] the loop and then do a streak in the third section. This
should start at the end of the streak in the second section.

7. Heat sterilize the loop again. Then, start the fourth line of culture
from the end of the third line and continue to the middle of the
plate. Be careful not to touch the previous lines.
Finally, heat the loop and label the plate. Place the plate upside
down in the incubator according to the instructions.
Other culture methods:

Radiant Streak method

Continuous streak method
Couture in broth medium
Tube-containing solid medium in shallow and deep culture
Culture in semi-solid medium
Antibiogram

Materials required for microbial

susceptibility testing:

Mueller Hinton Agar

Antimicrobial Discs

Mueller Hinton Agar medium should be checked for pH

after preparation at each stage of preparation.
The pH of the agar medium after gelation at room
temperature should be 7.4-7.2.

The diameter of the culture medium should be 4 mm.

Antimicrobial discs (storage):

The following conditions must be observed when storing

the discs:
Disks should be stored in a refrigerator (temperature 8°C
or lower) or in a freezer (-14°C or lower) until expiration.
Disks should not be stored in freezers with the ability to
defrost spontaneously.

Instructions for preparing standard 0.5McFarland:

[Link] 0.5 ml of 0.048 mol/l barium chloride (BaCl2) (2H2O
.W/VBaCl2 1.175%) to 5.99 ml of 0.18 mol/l sulfuric acid (1% V/V)
and obtain a suspension by stirring continuously.

[Link] the correct density of the standard turbidity by measuring

the absorbance in a spectrophotometer with a light path length of 1 cm.
The absorbance at 625 nm should be between 0.08 and 0.13.

3) The barium sulfate suspension should be poured into screw-capped

tubes of the same size as the bacterial suspension tubes in an amount of
6-4 ml.

Preparation of microbial suspension

Preparation of suspension by growth of bacteria in

liquid medium:
[Link] 3 to 5 completely isolated colonies of the same shape. Using
a loop or swab, pick off their tops and add to a tube containing 4-5
ml of liquid medium such as TSB.
[Link] the inoculated liquid medium in an incubator at 35°C until it
reaches a concentration equal to or greater than 0.5 McFarland
(usually 2-6 hours).
[Link] the turbidity of the liquid medium containing the growing
bacteria to the standard turbidity of 0.5 McFarland using liquid
culture medium or sterile physiological serum.
Inoculation of bacteria into the plate

[Link] 15 minutes of adjusting the turbidity of the bacterial suspension

to 0.5 McFarland standard, the bacteria should be inoculated into the
culture medium.
To do this, dip a swab into the tube containing the bacterial suspension,
rotate it several times, and press it against the inner wall of the tube
above the liquid level to remove excess liquid.

[Link] swab should be drawn over the surface of the Mueller Hinton
agar. This should be repeated 2 more times (3 times in total), rotating the
plate 60 degrees each time to ensure even distribution of the suspension
over the surface of the plate.
Finally, the swab should be drawn once around the outer edge of the
agar near the inner wall of the plate.
After inoculation and before discing, leave the plate lid ajar for 3-5
minutes to allow excess moisture to evaporate (maximum 15 minutes).
Note: To prevent inoculation of high bacterial concentrations, never use
overnight suspensions or suspensions prepared by non-standard
methods.

Placing a disc on a plate inoculated with

bacteria
[Link] discs for each bacterium should be placed carefully and
evenly on the Mueller Hinton agar plate with forceps and ensure complete
contact with the agar surface with slight pressure.
[Link] distance between the discs from center to center should not be less
than 24 mm. Also, too close to the edge of the plate will cause asymmetry
of the zone of inhibition (in some drugs).
3. The maximum acceptable number of discs on a plate with a diameter
of 150 mm is 12 and on a plate with a diameter of 100 mm, 6.
In all cases, it is better to place discs with a smaller diameter of the zone
of inhibition (gentamicin, vancomycin) next to discs with a larger
diameter of the zone of inhibition (cephalosporins) to reduce the
possibility of overlapping zones.

After 18-24 hours of incubation (except in exceptional cases), examine

each Petri dish.
If the plate is properly inoculated and the inoculation rate is correct, the
diameter of the zone of inhibition will be a uniform circle and the growth
will be dense and uniform.
If colonies appear singly, this indicates that the medium is too dilute and
the test should be repeated. Measure the diameter of the complete zones
of inhibition, including the diameter of the antibiotic disk, with the naked
eye.
Measure the diameter of the zone of inhibition from the back of the Petri
dish with a ruler while holding it a few centimeters above a matte black
surface and in the presence of reflected light. The following are
exceptions:(CLSI references)
Interpretation of disk diffusion test results
(interpretation criteria):

Susceptible: A group of isolated bacteria whose growth is inhibited

by the concentration of antimicrobial drug, based on the
recommended dose, usually obtained at the site of infection.
Intermediate: A group of isolated bacteria that respond less than
susceptible bacteria with the MIC level obtained in blood and tissue.
Resistant: A group of bacteria whose growth is not inhibited by the
usual concentrations of the drug at the usual prescribed dose or
whose growth inhibition zone is due to specific mechanisms of
microbial resistance .