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MICROBIOLOGY LABORATORY 9,10,11,12 (review)USTMED ’07 Sec C AsM; Photos provided by JV.N & MeaM.
DEMONSTRATIONS: MICROSCOPIC MORPHOLOGY OF DIFFERENT MICROORGANIMSMS
Sputum smear stained with Gram’s stain shows neutrophils, amorphous debris, and filamentous, beaded, branched gram-positive bacilli (oil immersion).
Gram stain of Bacillus cereus. The arrow is pointed at a spore, which is clear inside the gram-positive vegetative cell.
Escherichia coli.Stain used: Gram stainGram rxn: Gram negative (red)Morphology: Coccobacilli arranged singly or randomNote: E. coli is a Gram Negative Bacilli but it appears as a short plump bacilli so it is called coccobacilli.
Bacillus subtilis.Stain used: Gram StainGram rxn: Gram PositiveMorphology: Bacilli in chain (violet rods in chain). Aerobic sporeformer bacilli.Note: spores elliptical and centrally located
spores – unstained vegetative portion –
violet
Pseudomonas aeruginosaStain used: Gram StainGram rxn: Gram Negative bacilli in singly/random (red slender rods in singly or random)
The Gram staining method
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 Gram-positive.
- It’s a mystery - Although it may seem strange, the reason why
bacteria with these two major types of bacteria cell walls react differently with Gram’s stain appears to be unconnected with the wall structure itself. The exact method of the staining reaction is not fully 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, when dry, heated for a few seconds to the point when the glass slide is too hot to handle. This fixes ie kills the bacteria making the slide safe to handle. Care must be taken not to overheat which will char the cells.
4. Once cool, the slide is transferred to a support over a sink and flooded with a stain called Gentian Violet (a dye consisting of a methyl derivative of pararosaniline). The stain is left on the slide for about 1 minute. This stains all the bacteria on the slide a dark purple colour. Note, this stain will not penetrate the waxy cell walls of some 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 alcohol which causes the dye-iodine complex to be washed 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 which stains the Gram-negative cells a colour which contrasts markedly with the blue-black colour of the gram-posiitve cells. The stain common used for this is either eosin or fuchsin, both of which are red. These are called counterstains. Bacteria in the smear which are Gram-positive are unaffected by the counterstain.
9. The counterstain is left on the smear for about 30-60 seconds and then gently rinsed away with running water.
10. After the counterstain has been rinsed off, the slide is placed between some absorbent paper and the excess water gently blotted off. Care must be taken not to rub the slide with the blotting paper because this would remove the adhering bacteria.
11. The slide is gently warmed to dry off any residual moisture and then a drop of oil immersion oil is placed on the stained bacterial smear. This helps transmit light through the specimen directly to the high-powered microscope lens.
12. The slide is the placed on a microscope stage and the oil-immersion lens lowered into the immersion oil. High-powered lenses are required because bacteria are very small (hindi nga???!!!)
The resultsGram positive Gram negative
Staphylococcus aureusTypical Gram-positive
cocci in clusters
Escherichia coliTypical Gram-negative
coccobacilli, singly
Capsule stain. The cell is the purple rod in the center of the clear area. The purple color is from the basic stain, crystal violet.
The clear area is the capsule, and the background is colored by the negative, acidic stain (India ink).
Loefflers methylene blue stain.Corynebacterium diptheriae demonstrate metachromatic granules when stained with Loeffler methylene blue stain or Neisser stain. The stain is best performed on colonies grown on a Loeffler agar slant. Metachromatic deposits are reddish purple in Loeffler methylene blue stain.
Flagella stain.
Proteus sp. Peritrichous flagella – flagella distributed over the entire surface.
Vibrio cholera Monotrichous flagella – one polar flagellum.
Spore stain Bacillus cereus. The arrows are pointed at green spores in a pink vegetative cell.
Culture of Microorganisms
Different Streaking Methods
Culture Media Used: Eosin Methylene BlueOrganisms Used: Escherichia ColiMethod of Streaking: Simple Streaking
Illustration:
Culture Media Used: Eosin Methylene BlueOrganisms Used: Escherichia ColiMethod of Streaking: 4 Quadrant Method of Streaking
Note: This method of streaking is used for better isolation of the organism
Illustration:
Culture Media Used: Eosin Methylene BlueOrganisms Used: Escherichia ColiMethod of Streaking: Overlapping method of streaking
Note: This method of streaking is used for sensitivity testing
Illustration:
Antibiotic Sensitivity Testing Using The Kirby-Bauer Procedure
Kirby Bauer method. The test, introduced by William Kirby and Alfred Bauer in 1966, consists of exposing a newly-seeded lawn of the bacterium to be tested, growing on a nutrient medium (Mueller-Hinton agar) to filter paper disks impregnated with various antibiotics. The culture is incubated for 16 to 18 hours and then examined for growth. If the organism is inhibited by one of the antibiotics, there will be a zone of inhibition around the disk, representing the area in which the organism was inhibited by that antibiotic.
The diameter of the zone of inhibition around an antibiotic disk is an indication of the sensitivity of the tested microorganism to that antibiotic. The diameter of the zone, however, is also related to the rate of diffusion of the antibiotic in the medium. This fact must be kept in mind then interpreting the zone of inhibition of various antibiotics.
MATERIALS:- culture of the organisms in Mueller-Hinton agar
plate with antibiotic sensitivity discs- Ruler graduated in millimeters
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 provided3. Indicate if the antibiotics discs used is Sensitive,
Intermediate or Resistant.
Disk Diffusion Method
Procedure1. 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. Place the appropriate antimicrobial (sensitivity) discs using the dispenser or a sterile forceps. Incubate for 24 hours at 37o
3. Reading and Interpretation of the results. Measure the diameter of the Zone of Inhibition (area wherein there is no growth around the discs) using the millimeter of a ruler. Record your results and Interpret based on the table provided. Determine if the Antibiotic (organism) is 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.
Pseudomonas aeruginosa, a resistant strain. Growth on this Mueller-Hinton agar plate indicates that the isolate is resistant to six of 12 antimicrobial agents and susceptible to 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, CZ, CTX, CXM, CF, GM, NN: the three discs in the center of the plate are SXT, FOX and TIM.
Єtest – The Problem Solver in Antimicrobial Susceptibility Testing
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 and reproducible
Minimum Inhibitory Concentration (MIC) is generated for reliable
guidance of antimicrobial therapy.
Єtest Susceptibility Testing
Procedure1. inoculation of the
organism for testing using cotton swab by overlapping streaking using Mueller Hinton Agar Plate.
2. Overlaying of Єtest strip on the previously inoculated culture media
3. Reading of the strip and recording the results
A/N: … looks familiar right?! the next pictures are just recaps of our previous laboratory exercises. I’ll just list down the stuff that was brought out again for the review..
Review…1. Optochin sensitivity of
pneumococci…
2. Growth of pneumococci on blood agar showing draughtsman colonies…
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 stain16. Photochromogen M. kansasii17. MTb on LJ 8 weeks18. MTb on Middlebrook19. Mtb on middlebrook cording20. Gram stain of clostridium21. Gram stain of clostridium paraputrificum. Terminal
swollen spores22. Gram stain of Bacillus spp.23. Clostridium difficile, lecithinase24. Gram’s stain of a smear of exudates, gas bubbles25. Clostridia on egg yolk agar26. Clostridium tetati on Brucella blood agar27. Clostridium perfringens on brucella28. Gelatin hydrolysis test29. Growth of bacillus spp on egg yolk agar30. colonies of bacillus spp on 5% sheep blood agar31. sputum stained w/ gram’s stain shown w/ many
neutrophils32. colonies of c. diphtheriae on tinsdale agar33. colonies of c. diptheriae on 5% sheep blood agar34. Erysipelothrix on TSI agar35. colonies of erysipelothrix rhusiopathiae36. Esculin hydrolysis test37. colonies of listeria monocytogenes on 5% sheep
blood agar38. colonies of bacteroides fragilis on bile esculin39. colonies of bacteroides on brucella40. Gomori methenamine silver stain of actinomyces
spp41. Molar tooth appearance of actinomyces42. Amino acid hydrolysis rxn of nocardia asteroids43. colonies of nocardia asteroids growing on bay
plate44. modified kinyoun acid fast. Nocardia45. amino acid hydrolysis rxn of streptomyces46. gram stain of streptomyces47. gram stain of nocardia48. identification of the genus nocardia w/ biochemical
rxns
