MICROBIOLOGY LABORATORY 9,10,11,12 (review) USTMED ’07 Sec C AsM; Photos provided by JV.N & MeaM. 4.

DEMONSTRATIONS: MICROSCOPIC MORPHOLOGY Sputum smear stained with Gram’s stain shows neutrophils, amorphous debris, and filamentous, beaded, branched gram-positive bacilli (oil immersion). 5.


making the slide safe to handle. Care must be taken not to overheat which will char the cells. 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.

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 stain Gram rxn: Gram negative (red) Morphology: Coccobacilli arranged singly or random Note: 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 Stain Gram rxn: Gram Positive Morphology: Bacilli in chain (violet rods in chain). Aerobic sporeformer bacilli. Note: spores elliptical and centrally located • spores – unstained • vegetative portion – violet Pseudomonas aeruginosa Stain used: Gram Stain Gram 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. 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. 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

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 Grampositive. However wi would not be able to see those which had lost the dye-iodine complex which are called Gramnegative. 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 Grampositive 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 oilimmersion lens lowered into the immersion oil. Highpowered lenses are required because bacteria are very small (hindi nga???!!!) The results Gram positive Gram negative Staphylococcus aureus Escherichia coli Typical Gram-positive cocci in Typical Gram-negative clusters 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.

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: 1. 2. 3. The complete procedure is in you(r) lab manual. Measure the zone of inhibition and record your results. Interpret the results based on the table provided Indicate if the antibiotics discs used is Sensitive, Intermediate or Resistant.

Vibrio cholera Monotrichous flagella – one polar flagellum.

Spore stain Bacillus cereus. The arrows are pointed at green spores in a pink vegetative cell.

Disk Diffusion Method Procedure 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. 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.

Culture of Microorganisms Different Streaking Methods Culture Media Used: Eosin Methylene Blue Organisms Used: Escherichia Coli Method of Streaking: Simple Streaking Illustration:

Culture Media Used: Eosin Methylene Blue Organisms Used: Escherichia Coli Method of Streaking: 4 Quadrant Method of Streaking Note: This method of streaking is used for better isolation of the organism Illustration: 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 Procedure 1. inoculation of the organism for testing using cotton swab by overlapping streaking using Mueller Hinton Agar Plate.

Culture Media Used: Eosin Methylene Blue Organisms Used: Escherichia Coli Method 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


Overlaying of Єtest strip on the previously inoculated culture media Reading of the strip and recording the results


25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

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…


40. 41. 42. 43. 44. 45. 46. 47. 48.


Optochin sensitivity of pneumococci…


Growth of pneumococci on blood agar showing draughtsman colonies… Catalase test]

Clostridia on egg yolk agar Clostridium tetati on Brucella blood agar Clostridium perfringens on brucella Gelatin hydrolysis test Growth of bacillus spp on egg yolk agar colonies of bacillus spp on 5% sheep blood agar sputum stained w/ gram’s stain shown w/ many neutrophils colonies of c. diphtheriae on tinsdale agar colonies of c. diptheriae on 5% sheep blood agar Erysipelothrix on TSI agar colonies of erysipelothrix rhusiopathiae Esculin hydrolysis test colonies of listeria monocytogenes on 5% sheep blood agar colonies of bacteroides fragilis on bile esculin colonies of bacteroides on brucella Gomori methenamine silver stain of actinomyces spp Molar tooth appearance of actinomyces Amino acid hydrolysis rxn of nocardia asteroids colonies of nocardia asteroids growing on bay plate modified kinyoun acid fast. Nocardia amino acid hydrolysis rxn of streptomyces gram stain of streptomyces gram stain of nocardia identification of the genus nocardia w/ biochemical rxns


ustmedc3@yahoogroups.com audrey_cl@yahoo.com


Slide and tube coagulase tests


Mannitol Salt Agar (MSA)..S. aureus


Staph aureus on Blood Agar plate (BAP)


gram stain of staph aureus


Neisseria discharge..




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. 16. 17. 18. 19. 20. KInyoun’s acid fast stain Photochromogen M. kansasii MTb on LJ 8 weeks MTb on Middlebrook Mtb on middlebrook cording 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

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