MICROBIOLOGY LABORATORY 9,10,11,12 (review) making the slide safe to handle.

Care must be taken not to

USTMED ’07 Sec C AsM; Photos provided by JV.N & MeaM. overheat which will char the cells.
4. Once cool, the slide is transferred to a
DEMONSTRATIONS: MICROSCOPIC MORPHOLOGY OF DIFFERENT MICROORGANIMSMS support over a sink and flooded with a stain
called Gentian Violet (a dye consisting of a
Sputum smear stained with methyl derivative of pararosaniline). The
Gram’s stain shows neutrophils, stain is left on the slide for about 1 minute.
amorphous debris, and This stains all the bacteria on the slide a
filamentous, beaded, branched dark purple colour. Note, this stain will not
gram-positive bacilli (oil penetrate the waxy cell walls of some
immersion). bacteria eg mycobacteria.

5. The Gentian Violet is gently washed off the slide with running
water
6. The bacterial smear is then treated with Gram’s solution
which consists of 1 part iodine, 2 parts potassium iodide, and
300 parts water. This iodine solution reacts with the Gentian
Violet turning it a very dark shade of blue. It also causes it
to be retained by certain types of bacteria in a way which is
not really understood.
7. After about 30 seconds the slide is gently rinsed with ethyl
Gram stain of Bacillus cereus. The arrow is pointed at a spore, alcohol which causes the dye-iodine complex to be washed
which is clear inside the gram-positive vegetative cell. out of some bacteria but not others. This is called
decolourisation.
If we now look at the smear down a microscope, the
bacteria which had retained the Gentian Violet-iodine
complex will appear blue-black. These are called Gram-
positive. However wi would not be able to see those which
had lost the dye-iodine complex which are called Gram-
negative. The final step in the gram stain method is,
therefore, to stain the Gram-negative cells so they can be
seen.
8. This is achieved by treating the smear with a compound
Escherichia coli. which stains the Gram-negative cells a colour which contrasts
Stain used: Gram stain markedly with the blue-black colour of the gram-posiitve
Gram rxn: Gram negative (red) cells. The stain common used for this is either eosin or
Morphology: Coccobacilli arranged singly or random fuchsin, both of which are red. These are called
Note: E. coli is a Gram Negative Bacilli but it appears as a short counterstains. Bacteria in the smear which are Gram-
plump bacilli so it is called coccobacilli. positive are unaffected by the counterstain.
9. The counterstain is left on the smear for about 30-60 seconds
Bacillus subtilis. and then gently rinsed away with running water.
Stain used: Gram Stain 10. After the counterstain has been rinsed off, the slide is placed
Gram rxn: Gram Positive between some absorbent paper and the excess water gently
Morphology: Bacilli in chain blotted off. Care must be taken not to rub the slide with the
(violet rods in chain). Aerobic blotting paper because this would remove the adhering
sporeformer bacilli. bacteria.
Note: spores elliptical and 11. The slide is gently warmed to dry off any residual moisture
centrally located and then a drop of oil immersion oil is placed on the stained
• spores – unstained bacterial smear. This helps transmit light through the
• vegetative portion – violet specimen directly to the high-powered microscope lens.
12. The slide is the placed on a microscope stage and the oil-
Pseudomonas aeruginosa immersion lens lowered into the immersion oil. High-
Stain used: Gram Stain powered lenses are required because bacteria are very small
Gram rxn: Gram Negative bacilli in (hindi nga???!!!)
singly/random (red slender rods in
singly or random) The results
Gram positive Gram negative
Staphylococcus aureus Escherichia coli
The Gram staining method Typical Gram-positive cocci in Typical Gram-negative
clusters coccobacilli, singly
Gram’s Stain is a widely used method of staining bacteria as
an aid to their identification. It was originally devised by Hans
Christian Joachim Gram, a Danish doctor.
Gram’s stain differentiates between two major cell wall
types. Bacterial species with walls containing small amounts of
peptidoglycan and, characteristically, lipopolysaccharide, are
Gram-negative whereas bacteria w/ walls containing reltively
large amounts of peptidoglycan and no lipopolysaccharide are Capsule stain.
Gram-positive. The cell is the purple rod in the center of
the clear area. The purple color is from the
- It’s a mystery - basic stain, crystal violet.
Although it may seem strange, the reason why bacteria with
these two major types of bacteria cell walls react differently with The clear area is the capsule, and the
Gram’s stain appears to be unconnected with the wall structure background is colored by the negative,
itself. The exact method of the staining reaction is not fully acidic stain (India ink).
understood, however, this does not detract from its usefulness.

1. A small sample of a bacterial culture is removed from a

culture. In this example it is being taken from a broth culture
of the pure microbe but it could be removed from a culture
on solid medium or from material containing bacteria eg
faeces or soil.
2. The bacterial suspension is smeared onto a clean glass slide.
If the bacteria have been removed from a culture on solid
media or it is from a soil or faeces sample it will have to be
mixed with a drop of bacteria-free saline solution.
3. The bacterial smear is then dried slowly at first and the, Loefflers methylene blue stain.
when dry, heated for a few seconds to the point when the Corynebacterium diptheriae demonstrate metachromatic granules
glass slide is too hot to handle. This fixes ie kills the bacteria when stained with Loeffler methylene blue stain or Neisser stain.
The stain is best performed on colonies grown on a Loeffler agar must be kept in mind then interpreting the zone of inhibition of
slant. Metachromatic deposits are reddish purple in Loeffler various antibiotics.
methylene blue stain.
MATERIALS:
Flagella stain. - culture of the organisms in Mueller-Hinton agar plate
with antibiotic sensitivity discs
Proteus sp. Peritrichous flagella – - Ruler graduated in millimeters
flagella distributed over the entire
surface. NOTE:
- The complete procedure is in you(r) lab manual.
1. Measure the zone of inhibition and record your results.
2. Interpret the results based on the table provided
Vibrio cholera Monotrichous flagella – one polar flagellum. 3. Indicate if the antibiotics discs used is Sensitive,
Intermediate or Resistant.

Spore stain Disk Diffusion Method

Bacillus cereus. The arrows are
pointed at green spores in a pink Procedure
vegetative cell. 1. dip the sterile swab into bacterial suspension compared
to 0.5 MF standard then swab onto the surface of
Mueller Hinton Agar using Overlapping technique.
2. Allow the organism to be absorbed by the medium.
Culture of Microorganisms Place the appropriate antimicrobial (sensitivity) discs
using the dispenser or a sterile forceps. Incubate for 24
Different Streaking Methods hours at 37o
3. Reading and Interpretation of the results. Measure the
Culture Media Used: Eosin Methylene Blue diameter of the Zone of Inhibition (area wherein there is
Organisms Used: Escherichia Coli no growth around the discs) using the millimeter of a
Method of Streaking: Simple Streaking ruler. Record your results and Interpret based on the
table provided. Determine if the Antibiotic (organism) is
Illustration: Sensitive, Intermediate or Resistant. If there is
overlapping in the zone of inhibition, you can just
measure the radius and multiply the reading by 2 to get
the diameter.

Culture Media Used: Eosin Methylene Blue

Organisms Used: Escherichia Coli
Method of Streaking: 4 Quadrant Method of Streaking
Pseudomonas aeruginosa, a resistant strain.
Note: This method of streaking is used for better isolation of the
Growth on this Mueller-Hinton agar plate
organism
indicates that the isolate is resistant to six of
12 antimicrobial agents and susceptible to
Illustration:
the remaing. The isolate is resistant to SXT,
GM, ATM, TIM, TIC and MMZ. The isolate is
susceptible to CIP, AN, NN, CA, IPM and PIP.

E. coli ATCC 25922. The isolate tested on

this Mueller-Hinton agar plate is interpreted
as susceptible (S) to all antimicrobial agents.
Reading clockwise from the top, MZ, AN, AM,
Culture Media Used: Eosin Methylene Blue
CZ, CTX, CXM, CF, GM, NN: the three discs in
Organisms Used: Escherichia Coli
the center of the plate are SXT, FOX and TIM.
Method of Streaking: Overlapping method of streaking
Єtest – The Problem Solver in Antimicrobial
Note: This method of streaking is used for sensitivity testing
Susceptibility Testing
Illustration:
Etest is an antimicrobial
gradient strip for the
quantitative determination of
susceptibility or resistance of
microorganisms. It is a robust
and simple technique,
minimally affected by
laboratory variations and can
be used to test most
microorganisms. An accurate
Antibiotic Sensitivity Testing Using The Kirby-Bauer Procedure
and reproducible Minimum
Inhibitory Concentration (MIC)
Kirby Bauer method. The test, introduced by William Kirby and
is generated for reliable
Alfred Bauer in 1966, consists of exposing a newly-seeded lawn of
guidance of antimicrobial
the bacterium to be tested, growing on a nutrient medium
therapy.
(Mueller-Hinton agar) to filter paper disks impregnated with
various antibiotics. The culture is incubated for 16 to 18 hours
Єtest Susceptibility Testing
and then examined for growth. If the organism is inhibited by one
of the antibiotics, there will be a zone of inhibition around the
Procedure
disk, representing the area in which the organism was inhibited by
that antibiotic. 1. inoculation of the
organism for testing
The diameter of the zone of inhibition around an antibiotic disk is using cotton swab by
an indication of the sensitivity of the tested microorganism to that overlapping streaking
antibiotic. The diameter of the zone, however, is also related to using Mueller Hinton
the rate of diffusion of the antibiotic in the medium. This fact Agar Plate.
 2. Overlaying of Єtest 25. Clostridia on egg yolk agar
strip on the previously 26. Clostridium tetati on Brucella blood agar
inoculated culture 27. Clostridium perfringens on brucella
media 28. Gelatin hydrolysis test
29. Growth of bacillus spp on egg yolk agar
3. Reading of the strip and recording the results 30. colonies of bacillus spp on 5% sheep blood agar
31. sputum stained w/ gram’s stain shown w/ many
neutrophils
32. colonies of c. diphtheriae on tinsdale agar
33. colonies of c. diptheriae on 5% sheep blood agar
34. Erysipelothrix on TSI agar
35. colonies of erysipelothrix rhusiopathiae
36. Esculin hydrolysis test
37. colonies of listeria monocytogenes on 5% sheep blood
agar
38. colonies of bacteroides fragilis on bile esculin
A/N: … looks familiar right?!  the next pictures are just recaps
of our previous laboratory exercises. I’ll just list down the stuff
39. colonies of bacteroides on brucella
40. Gomori methenamine silver stain of actinomyces spp
that was brought out again for the review..
41. Molar tooth appearance of actinomyces
42. Amino acid hydrolysis rxn of nocardia asteroids
Review…
43. colonies of nocardia asteroids growing on bay plate
1. Optochin sensitivity of pneumococci… 44. modified kinyoun acid fast. Nocardia
45. amino acid hydrolysis rxn of streptomyces
46. gram stain of streptomyces
47. gram stain of nocardia
2. Growth of pneumococci on blood agar 48. identification of the genus nocardia w/ biochemical rxns
showing draughtsman colonies…
ustmedc3@yahoogroups.com
audrey_cl@yahoo.com
3. Catalase test]

4. Slide and tube coagulase tests

5. Mannitol Salt Agar (MSA)..S. aureus

6. Staph aureus on Blood Agar plate (BAP)

7. gram stain of staph aureus

8. Neisseria gonorrhea. Urethral

discharge..

9. Myco TB on LJ media

10. Scotochromogen M. gordonae

11. M. Tb fluorochrome stain

12. M Tb kinyoun

13. Kinyoun acid fast stain

14. MTB kinyoun

15. KInyoun’s acid fast stain

16. Photochromogen M. kansasii
17. MTb on LJ 8 weeks
18. MTb on Middlebrook
19. Mtb on middlebrook cording
20. Gram stain of clostridium
21. Gram stain of clostridium paraputrificum. Terminal
swollen spores
22. Gram stain of Bacillus spp.
23. Clostridium difficile, lecithinase
24. Gram’s stain of a smear of exudates, gas bubbles