BSCI 424 — PATHOGENIC MICROBIOLOGY — Fall 2000

Laboratory Exercises

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1. To provide practice in isolating, in pure culture, single microorganisms from a culture.
2. To review the Gram stain.
3. To provide instruction in microscopy required for observing bacteria on a routine basis.

• Staphylococcus aureus
• Escherichia coli
• ß-hemolytic streptococci
• Bacillus subtilis
• Corynebacterium xerosis
• Mixed culture

• Blood Agar Plates (BAP): for colony morphology and hemolysis
• Trypticase Soy Agar (TSA): to practice streaking technique for isolation; for colony morphology
• MacConkey agar: to distinguish lactose-fermenting gram-negative organisms from nonfermenters

• Streaking agar plates
• Gram stain
• Isolation of bacteria from skin
• Examine BAP for hemolysis
• Examine MacConkey plate for lactose-fermenting colonies and/or nonfermenters

1. To isolate pure cultures of bacteria from various parts of body in order to become acquainted with the "common microbiota" (normal flora) residing there and to practice the isolation of bacteria from collected specimens.
2. The ability to perform certain biochemical reactions is one of the criteria commonly employed to discriminate between different bacteria. Another is susceptibility to certain antibiotics (see Exercise #7) . You will learn how to perform the catalase, oxidase, coagulase and fibrinolysin tests. Positive controls are to be used in each experiment.
3. To observe and distinguish between types of hemolytic activity.

Cultures:        Go to Pathogen List

• Staphylococcus aureus
• Staphylococcus epidermidis
• Streptococcus pyogenes (Group A)
• Moraxella catarrhalis
• Finger isolates from Exercise #1

• Blood Agar (BAP): for isolation; to observe colony morphology and hemolysis

• Isolate microorganisms from the skin
• Gram Stain
• Catalase Test
• Oxidase Test
• Coagulase Test
• Fibrinolysin Test (Optional Demo only)
• Observe colony morphology and hemolysis

Objective: To differentiate pathogenic from non-pathogenic members of the family Micrococcaceae.

• Staphylococcus aureus
• Staphylococcus epidermidis
• Micrococcus luteus
• Unknown(s) for Each Group

• Coagulase Test Medium
• Blood Agar (BAP): determine hemolytic patterns
• Mannitol Salt Agar (MSA): for selective isolation of coagulase-positive, mannitol-fermenting Staphylococcus
• Phenylethyl Alcohol Agar (PEA): for the isolation of Staphylococcus and inhibition of gram-negative bacilli (particularly Proteus)
• Glucose Broth (overlaid with mineral oil after inoculation): for anaerobic fermentation
• Trypticase Soy Agar (TSA): for catalase test

Procedures:

• Inoculate media & observe growth and biochemical reactions
• Gram stain
• Glucose broth overlayed with sterile mineral oil
• b-lactam disk sensitivity
• Catalase Test
• Tube Coagulase Test
• Slide Coagulase Test
• Isolate respiratory microorganisms from nasal swabs
• Observe hemolysis, colony morphology and pigment production 

Objective: To demonstrate the culture characteristics of certain species of streptococci.

• Streptococcus pyogenes (group A)
• Streptococcus agalactiae (group B)
• Enterococcus faecalis
• Streptococcus pneumoniae
• Unknown(s) for Each Group

• Blood Agar (BAP): test for aerobic and anaerobic hemolysis; CAMP TEST.
• Bile Esculin Agar (BEA): for detection of fecal streptococci (group D) and enterococci; test for hydrolysis of esculin to esculetin
• SF Broth (Streptococcus [Enterococcus] faecalis broth): for detection of fecal streptococci (group D) and enterococci; test for fermentation of glucose
• Trypticase Soy Agar (TSA): for catalase test

Procedures:

• Inoculate media & observe growth and biochemical reactions
  • BEA plate: ability to grow in presence of bile acids; esculin hydrolysis
  • SF broth: glucose fermentation
• Gram stain
• Taxo A (bacitracin) disk sensitivity
• Taxo P (optochin) disk sensitivity
• CAMP Test
• Catalase Test 

Objective: To understand the identifiable characteristics of members of the Corynebacteriaceae family when grown on specific media.

• C. diphtheriae
• C. xerosis
• C. pseudodiphtheriticum
• Unknown(s) for Each Group

• Cystine Tellurite Blood Agar: differential and selective isolation of C. diphtheriae
• Cystine Trypticase Agar (CTA): to detect fermentation of supplemented carbohydrates; for detemination of motility
  • CTA glucose
  • CTA sucrose
    

Procedures:

• Inoculate media & observe growth and biochemical reactions
• Gram stain
• Methylene Blue Stain (Toluidine blue O homologue)  

Objective: To acquaint you with the biochemical tests which are routinely used in the identification of the genera and species in the family Enterobacteriaceae.

• Arizona hinshawii
• Morganella morganii
• Citrobacter freundii
• Proteus mirabilis
• Proteus vulgaris
• Edwardsiella spp.
• Providencia rettgeri
• Enterobacter aerogenes
• Salmonella paratyphi
• Salmonella spp. Group B
• Escherichia coli
• Serratia marcescens
• Klebsiella pneumoniae
• Unknown(s) for Each Group
• Non-Enterobacteriaceae Controls: Alcaligenes faecalis; Pseudomonas aeruginosa

• Salmonella typhi
• Shigella spp.
• Yersinia pestis
• Yersinia enterocolitica

• Trypticase Soy Agar (TSA): for isolation of fingertip organism(s) for Exercise 7.
• Triple Sugar Iron Agar (TSI): for beginning the identification of a gram-negative enteric bacillus by testing for acid/alkaline reactions from glucose, lactose, sucrose and peptone nutritional sources; for testing reduction of sulfur and production of H₂S.
• Urease Broth or Urea Agar Slant: to test for urea hydrolysis
• Simmons Citrate Agar: to test for utilization of citrate as the sole source of carbon
• Motility-Indole-Ornithine Agar: to test for motility, decarboxylation of ornithine and indol production
• Lysine Decarboxylase Medium: to test for production of an amine by decarboxylation of lysine
• Sugar Utilization Media: to test for utilization of glucose, sucrose, mannitol, or lactose and to determine gas production from glucose
• MacConkey Agar: to differentiate gram-negative lactose fermenters from nonfermenters
• XLT-4 Agar: for selective and differential isolation of Salmonella and to test for reduction of sulfur to H₂S
• Brilliant Green Agar: for selective isolation of Salmonella, except S. typhi.

Procedures:

• Gram stain
• Inoculate media & observe growth and biochemical reactions
• Identify each organism 

1. To demonstrate methods to determine microbial resistance to antibiotics.
2. To determine the variability of antibiotic susceptibility of common microbiota.
3. To demonstrate how serum levels of antibiotics are measured.

• Staphylococcus aureus
• Skin isolate (unknown)

• Mueller-Hinton Broth: for broth tube antibiotic MIC assay
• Mueller-Hinton Agar: for disk diffusion antibiotic susceptibility, antibiotic serum level measurements and MBC determination

Procedures:

• Broth Tube MIC (Minimal Inhibitory Concentration)
• Disk-diffusion Method (Kirby-Bauer Method)
• Assay of serum levels of an antibiotic
• MBC (Minimal Bactericidal Concentration)

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