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Microbiology Lab Practical Review for MLAB352

This review covers biosafety rules, staining techniques, plate streaking, counting, media types, temperature and oxygen classifications, as well as controls. It includes guidelines for lab hygiene and specimen handling.

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Microbiology Lab Practical Review for MLAB352

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  1. Review for Lab Practical #1 MLAB 352 Dr. Mohamed A. El-Sakhawy

  2. Lecture Outline • Exam format • Biosafety • Staining • Four-way plate streak • Plate counts • Media types • Temperature classification • Oxygen classification • Control with chemicals and antibiotics

  3. Biosafety

  4. General Biosafety Rules • NO food or drink in lab. • NO cosmetics in lab. • NO open-toed shoes in lab. • Lab doors will remain closed at all times. • NO mouth pipetting. • Use care when handling potentially hazardous materials. • Wear appropriate personal protective equipment at all times. • Dispose of all microbiological materials in designated orange biohazard bags. • Dispose of all sharps and glass in designated sharps containers. • Notify the instructor immediately when there is an accident or spill. • Notify the instructor of pertinent medical conditions.

  5. When You Begin Work for the Day… • Wash your hands. • Disinfect the surface of your lab table. • Put on your goggles. • Put on your lab apron. • Put on gloves.

  6. When You Are Finished for the Day… • Appropriately dispose of all waste. • Disinfect the surface of your lab table. • Remove gloves inside-out and dispose in biohazard waste. • Remove apron, fold inside-out, and store in your drawer. • Remove goggles and store in your drawer. • Wash your hands before leaving the lab.

  7. Staining

  8. Types of Bacterial Stains • Simple stain – Stains cells but not background • Heat-fixed (killed) bacteria • Negative stain – Stains background but not cells • Live bacteria • Differential stain – Stains only certain types of cells based upon properties • Basic stain – Positive charge • Attracted to negatively-charged cell wall • Acidic stain – Negative charge • Repelled by negatively-charged cell wall

  9. Reagents of a Differential Stain • Primary stain • First dye used in the staining process • Will initially stain all cells and then be removed from a subset • Mordant • Improves the ability of the primary stain to bind cells • Decolorizer • Removes the primary stain from a subset of cells • Counterstain • Second dye that stains decolorized cells

  10. A Smear is Prepared by Spreading Bacteria on a Glass Slide Figure 4.15

  11. Heat-Fixing a Bacterial Smear http://iws2.collin.edu/dcain/CCCCD%20Micro/Heat_fix.htm

  12. Gram Stain (primary stain) (mordant) (decolorizer) (counterstain) http://iws2.collin.edu/dcain/CCCCD%20Micro/gram_stain.htm http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/grmix.html

  13. Examples of Gram-Positive and Gram-Negative Organisms GRAM-POSITIVE GRAM-NEGATIVE Escherichia coli • Staphylococcus xylosus • Bacillus megaterium

  14. Capsule Stain NO HEAT FIXATION! • Alcaligenes faecalis • Enterobacter aerogenes • Klebsiella pneumoniae • Serratia marcescens (primary stain) (decolorizer/counterstain) http://iws2.collin.edu/dcain/CCCCD%20Micro/capsule_stain.htm

  15. Acid-Fast Stain (primary stain) (decolorizer) (counterstain) • Mycobacterium smegmatis Figure 4.18 http://iws2.collin.edu/dcain/CCCCD%20Micro/acid_fast.htm

  16. Endospore Stain (primary stain) (decolorizer) (counterstain) • Bacillus megaterium http://iws2.collin.edu/dcain/CCCCD%20Micro/endospore.htm

  17. Four-Way Plate Streak

  18. Four-Way Plate Streak http://iws2.collin.edu/dcain/4way.htm

  19. Plate Counts

  20. Viable Plate Counts from Serial Dilutions • TNTC – Too Numerous To Count • More than 300 colonies • TFTC – Too Few To Count • Less than 30 colonies

  21. Calculation of CFU/ml for Urine Specimens • Count colonies • CFU/ml = # colonies/calibrated loop volume • Threshold: 1 x 105 CFU/ml

  22. Media Types

  23. Nutrient Agar (NA) • Special features: • None • Used for: • Culture of non-fastidious organisms http://commons.wikimedia.org/wiki/File:Agar_Plate.jpg

  24. Sabouraud Agar/Broth • Selective media • Special features: • Acidic pH (5.5) • Used for: • Culture of fungi (yeasts and molds) http://iws2.collin.edu/dcain/CCCCD%20Micro/sabouraudagar.htm http://www.jlindquist.net/generalmicro/dfnewgfbpage.html

  25. Brain Heart Infusion (BHI) Agar • Special features: • None • Used for: • BHI agar deeps used to determine oxygen classification http://iws2.collin.edu/dcain/CCCCD%20Micro/bhitubes.htm

  26. Sodium Thioglycollate Broth • Special features: • Sodium thioglycollate – Reduces O2 to H2O • Resazurin (dye) – Pink when oxidized (O2 present), colorless when reduced (no O2) • Used for: • Culture of anaerobic organisms http://iws2.collin.edu/dcain/CCCCD%20Micro/thioglycollatebroth.htm

  27. Mannitol Salt Agar (MSA) • Selective • Differential • Special features: • 7.5% NaCl • Mannitol (fermentation substrate) • Phenol red (pH indicator) • Used for: • Growth of halotolerant species (esp. Staphylococcus spp.) • Distinguishing between Staphylococcus spp. S. epidermis S. aureus/ S. xylosus http://iws2.collin.edu/dcain/CCCCD%20Micro/normalskinflora.htm

  28. Blood Agar • Differential • Enriched • Special features: • Sheep’s blood (nutrients, hemolysis substrate) • Used for: • Culture of fastidious organisms • Distinguishing between bacteria by hemolysis patterns (esp. Streptococcus) http://iws2.collin.edu/dcain/CCCCD%20Micro/hemolysis.htm

  29. Chocolate Agar • Enriched • Special features: • Boiled sheep’s blood (nutrients) • Used for: • Growth of fastidious organisms (esp. Neisseria spp.) • Distinguish Neisseria spp. using oxidase test http://iws2.collin.edu/dcain/CCCCD%20Micro/normalfloranasopharynx.htm http://www.medschool.lsuhsc.edu/microbiology/DMIP/bsoxid.jpg

  30. Mueller-Hinton Tellurite (MHT) Agar • Differential • Special features: • Tellurite (metal) • Used for: • Distinguishing Corynebacterium spp. (diphtheroids) based on reduction of tellurite to tellurium (black ppt.) http://iws2.collin.edu/dcain/CCCCD%20Micro/normalskinflora.htm

  31. Columbia C-NA (CCNA) Agar • Selective • Differential • Special features: • Colistin (antibiotic) • Nalidixic acid (antibiotic) • Sheep’s blood (nutrients/hemolysis substrate) • Used for: • Selective culture of Gram-positive species • Distinguishing between Gram-positives based on hemolysis E. aerogenes E. faecalis E. coli S. xylosus http://iws2.collin.edu/dcain/CCCCD%20Micro/columbiacnaagar.htm

  32. MacConkey Agar • Selective • Differential • Special features: • Crystal violet (dye) • Bile salts • Lactose (fermentation substrate) • Neutral red (pH indicator) • Used for: • Selective culture of Gram-negative organisms • Distinguishing between Gram-negatives based on lactose fermentation E. aerogenes E. coli S. epidermis S. typhimurium http://iws2.collin.edu/dcain/CCCCD%20Micro/macconkeyagar.htm

  33. Eosin Methylene Blue (EMB) Agar • Selective • Differential • Special features: • Eosin (dye) • Methylene blue (dye) • Lactose (fermentation substrate) • Used for: • Selective culture of Gram-negative species • Distinguishing between Gram-negatives based on lactose fermentation P. aeruginosa E. coli S. xylosus E. aerogenes http://iws2.collin.edu/dcain/CCCCD%20Micro/embagar.htm

  34. Snyder Test Agar • Selective • Special features: • Acidic pH (4.8) • 2% glucose (fermentation substrate) • Bromcresol green (pH indicator) • Used for: • Assessing levels of Lactobacillus spp. organisms in the mouth • Testing susceptibility to dental caries http://iws2.collin.edu/dcain/CCCCD%20Micro/snydertest.htm

  35. Temperature Classification

  36. All Microbes Have an Optimal Growth Temperature Psychrotrophs Figure 6.5

  37. Incubation at Multiple Temperatures Identifies Temperature Classification Figure 6.4b

  38. Examples of Temperature Classifications http://iws2.collin.edu/dcain/CCCCD%20Micro/temperatureclassification.htm

  39. Oxygen Classification

  40. BHI Agar • Obligate aerobe • Microaerophile • Facultative anaerobe • Aerotolerant anaerobe • Obligate anaerobe http://iws2.collin.edu/dcain/CCCCD%20Micro/bhitubes.htm

  41. Sodium Thioglycollate Broth • Obligate anaerobe • Obligate aerobe • Aerotolerant anaerobe • Facultative anaerobe http://iws2.collin.edu/dcain/CCCCD%20Micro/thioglycollatebroth.htm

  42. Control with Chemicals and Antibiotics

  43. The Disk Diffusion Assay Measures Sensitivity Figure 10.9

  44. A Susceptibility Chart is Used to Determine Effects of Antibiotics

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