Microbio Lab 6

MICROBIOLOGY LABORATORY 6 USTMED ’07 Sec C – AsM; Photos provided by JV.N & MeaM. Amino acid hydrolysis reactions of Nocardia asteroids. All amino acid reactions are negative on this identification plate: casein, tyrosine, xanthine and hypoxanthine.

Colonies of Erysipelothrix rhusiopathiae. Colonies are smooth, small (less than 1.0mm in diameter), and transparent. A greenish color and/or α-hemolysis appears as the colonies age.

a Colonies of Nocardia asteroides on 5% sheep blood agar. Colonies initially appear as powdery, and as they mature they can develop a number of colors, ranging from a chalky-white to yellow-orange, buff, red, purple, brown and black. Most colonies have an earthy odor.

Colonies of Nocardia asteroids growing on BAY late (x15). Colonies are large, dry, bumpy, and heaped after 9 days incubation at 27oC. This morphology has been referred to as glabrous orange form.

Identification of the genus Nocardia with biochemical reactions. Nocardia spp. are aerobic actinomycetes that reduce nitrate to nitrite (tube on left), hydrolyze urea (tube in center), and grow on the surface of a broth medium, such as the thioglycolate broth on the right.

Erysipelothrix rhusiopathiae on tripe sugar iron (TSI) agar. Erysipelothrix rhusiopathiae demonstrates H2S production along the stab line in a TSI agar slant within 48 hours of incubation at 35oC. This is helpful feature that separates this organism from other gram-positive bacilli.

Amino acid hydrolysis reactions of Streptomyces spp. All reactions are positive on this identification plate. Unlike any Nocardia spp., Streptomyces spp. hydrolyze casein, tyrosine, xanthine, and hypoxanthine. Colonial morphology resembles the chalky, dry white colonies of Nocardia spp. Although Streptomyces spp. are urea-positive, they are nitrate-negative, distinguishing the genera from Nocardia spp. Molar tooth appearance of Actinomyces israelii after incubation for 1 week.

Gram stain of Streptomyces spp. (x1250). Streptomyces spp. are characterized microscopically by branching, beaded, gram-positive filamentous bacilli, indistinguishable from Nocardia spp. Unlike Nocardia spp. the partial acid-fast stain is negative.

Gomori methenamine silver stain of Actinomyces spp. (x1250). The slide was prepared from a tissue biopsy specimen. In the center is a characteristic “sulfur granule,” which consists of a granular microcolony surrounded by purulent xudate. The Actinomyces spp. are gram-postive bacilli that vary in size from short, diphtheroid forms to long, branching filaments. Unlike Nocardia spp. they are not partially acid-fast. Gram stain Nocardia spp. (x1250). Nocardia species are branching, beaded, filamentous gram-positive bacilli, approximately 1um in diameter. They can also appear as coccoid or coccobacillary forms. Care should be taken when examining the slides because of the faint staining properties of these microorganisms and other actinomycetes.

Skin punch biopsy with positive acid fast stain of Mycobacterium leprae.

Acid Fast Staining Materials for Acid Fast Staining

oun acid-fast st when stained as the o distinguish this .

Photochromogen M. kansasii colonies when exposed to light have strong yellow color. Colonies of M. tuberculosis growing on Middlebrook agar. Cording characteristics of M. tuberculosis. Kinyoun’s Carbol Fuchsin Acid Alcohol Methylene Blue Kinyoun’s acid fast stain (x1500). Mycobacteria are readily stained with carbol fuchsin, which binds the mycolic acid in their lipid-rich cell walls. This stain cannot be removed (decolorized) with acid alcohol and, therefore, the microorganisms are referred to as acid-fast. There are two common acid-fast stains, Ziehl-Neelsen (ZN), and Kinyoun’s. The difference is that the ZN stain requires heat during the staining process because the phenol concentration used is less than in the Kinyoun’s method. The decolorizer, acid alcohol, and the counterstain, methylene blue, are the same for both methods. When stained, acid-fast bacilli stain red and the background is blue, as shown here. M. Tuberculosis stained with fluorochrome stain.

M. tuberculosis stained with Kinyoun acid-fast stain.

Colonies of M. tuberculosis growing on Middlebrook agar. Scotochromogen M. gordonae with yellow colonies.

M. Tuberculosis colonices on Lowenstein-Jensen agar after 8

weeks of incubation.

Mycobacterium tuberculosis on Lowenstein Jensen medium (LJ) Mycobacterium tuberculosis colonies on Lowenstein-Jensen media. The organism grows in culture as slowly developing, rough colonies with a characteristic buff color. Under optimal conditions, colonies are recognizable in 3 weeks, but some strains may require 4 to 6 weeks. LJ media, an egg-based media containing malachite green to inhibit growth of contaminating bacteria, is commonly used in clinical laboratories. Other mycobacterial isolation media include Middlebrook 7H11, Middlebrook 7H10 (used predominantly for susceptibility testing), mycobactosel, and Middlebrook 7H9 broth. For optimal recovery, a combination of liquid and solid media is recommended. M. tuberculosis on LJ agar slant

Mycobacterium tuberculosis Stain used: Kinyoun’s Acid fast stain Clinical specimen used: Sputum Results: Positive for acid fast bacilli. acid fast stain of direct smear to show acid fast bacilli staining deep red (arrow A) and non-acid fast bacilli cells staining blue with the counter stain methylene blue (arrow B) stain. Nocardia species with a modified Kinyoun stain decolorizing agent. This mircroorganism from other

Mycobacterium tuberculosis Clinical specimen used: Sputum Stain used: kinyoun’s acid fast staining Acid Fast Bacilli appears as red bacilli (arrow A). The pus cells and the non-acid fast bacilli stained with counter stain which is the methylene blue (arrow B).

LJ agar has been used as the standard for isolating Mycobacteria. It contains coagulated whole eggs, glycerol, potato flour, and salts. Malachite green is added to inhibit the growth of contaminating bacteria. The disadvantage of this medium is that it becomes hydrolyzed when contaminants do grow on it, and the culture must be discarded. Illustrated here are rough, half-colored colonies that appeared w/in 3 weeks typical of M. tuberculosis. Colonies of Bacillus spp. on 5% sheep blood agar. Some species of Bacillus are nonhemolytic with mucoid and spreading colonies resembling the Enterobacteriaceae and Pseudomonas spp. Colonies of Bacillus anthracis, the major human pathogen, are usually nonhemolytic. This figure shows and environmental Bacillus sp. that rarely causes human infections. Gelatin hydrolysis test. Many Bacillus spp. secrete proteolytic enzymes that can hydrolyze gelatin. In the method shown in this figure, exposed, undeveloped x-ray film is used as the substrate for detection of gelatinase activity. Hydrolysis destroys the film’s gelatin coating, leaving only the clear photographic film where the strip was

immersed in the organism suspension.

Sputum smear stained with Gram’s stain shows neutrophils, amorphous debris, and filamentous, beaded, branched grampositive bacilli (oil immersion) Colonies of Bacillus spp. on 5% sheep blood agar are smooth, round, and surrounded by a zone of beta hemolysis.

Growth of Bacillus spp. on egg yolk agar. Reaction on egg yolk agar is one of the characteristics used to identify Bacillus spp. Bacillus cereus is one of many species that produce the enzyme lecithinase, demonstrated by a zone of opacity (whitish color in the agar) extending away from the bacterial growth, as shown on the left. Lipase activity can also be detected on egg yolk agar, demonstrated by an oily looking sheen on the surface of the bacterial growth but not extending onto the agar, as shown on the right.

Colonies of Bacteroides fragilis on Bacteroides bile esculin (BBE) agar. This medium contains gentamicin to inhibit facultative gramnegative bacilli. Bile salts inhibit most anaerobes other than those of the B. fragilis group, which grow as large, gray to black colonies due to their hydrolysis of esculin in the medium. Colonies of C. diphtheriae on Tinsdale agar. A selective medium should be used along with sheep blood agar whenever Coryebacterium diphtheriae is suspected, as well as to differentiate C. diphtheriae from other corynebacteriua. ON the selective medium cystine tellurite agar or potassium tellurite (Tinsdale) agar, the colonies of Corynebacterium diphtheriae have a gun-metal, gray-black appearance. Sputum stained with Gram’s stain shows many neutrophils, amorphous debris, and coryneform gram-positive bacilli (oil immersion)

Esculin hydrolysis test. Most strains of Listeria monocytogenes can hydrolyze esculin in less than 2 hours. This photograph shows positive reactions for bile esculin (tube on left) and hippurate (right).

Gram stain of Bacillus spp. Large gram positive bacilli with squared-off ends measuring 0.8 x 7.5 um. Occurring singly and in chains.

Corynebacterium diphtheriae

Clostridium botulinum Colonies of Bacteroides fragilis on Brucella blood agar. Bacteroides spp. as well as other anaerobic gram-negative bacilli grow best on this nonselective mediu. Agar must be supplemented with hemin and vitamin K1 to support the growth of many species of anaerobes. These colonies appear as circular, entire and raised. Colonies of Listeria monocytogenes on 5% sheep blood agar. Colonies of Listeria monocytogenes are small (less than 1mm in diameter), smooth, irregular, and translucent. The colonies are surrounded by a very characteristic narrow zone of beta hemolysis on 5% sheep blood agar and may be confused with group B beta-hemolytic streptococci. Differentiating characteristics include Listeria’s narrow zone of beta hemolysis, positive catalase production, and growth on bile and hydrolysis of esculin. Both species hydrolyze hippurate.

Colonies of C. diphtheriae on 5% sheep blood agar. Corynebacterium diphtheriae grow well on 5% sheep blood agar as whitish, opaque colonies

Reverse CAMP test for presumptive identification of Clostridium perfringens. C. perfringens is streaked verticall and Streptococcus agalactiae is streaked horizontally in this test. The hemolysis of the clostridium is synergistically enhanced by the hemolysin of the streptococcus in an arrowhead-shaped pattern. Clostridium tetani on Brucella blood agar. Growth on agar is characterized by swarming, such that growth will cover the entire plate in a thin film within a few days.

Clostridium perfringens on Brucella blood agar. Colonies display the double-zone of beta hemolysis typical of this pecies. Colonies are large with peaked centers and irregular edges after 48 hours incubation.

Clostridium difficile and C. perfringens on egg yolk agar. The C. perfringens (upper half of plate) produces abundant lecithinase, while the C. difficile (lower half) grows but shows no reaction. Growth of clostridia on egg yolk agar. The production of the enzymes lecithinase, opaque white precipitate extending from the colony into the medium right side), and lipase, iridescent sheen on surface of colony (left side) is used to differentiated among Clostridium species and to help identify some Fusobacterium species. Gram stain of Clostridium spp. (x1250). Cells are parallel-sided, long, thin, gram-variable, and some show swollen ends indicative of spore formation. Gram stain of Clostridium paraputrificum (x1250). This microorganism displays terminal, swollen spores and the gram-variable staining typical of Clostridium species. Spores may not take up the stain so they may appear as clear areas. [email protected] [email protected] c3, thank you talaga for the support! Pagpasensyahan niyo na ‘to, I know this could have been helpful for the first practicals.. didn’t realize kasi na kasama sya sa scope.. anyway, enjoy na lang the pics… jayveeh, ate mea, thanks for the digital pics, wala ako nagagawang notes without them! Leigh, milo, anne, thanks sa tulong! Bait niyo talaga! eto na siguro yung hardest notes na ginawa ko, sobrang daming pictures!!!! I hate making lab notes! Argggghhhh!!! -auds-