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. Format of the Lab Practical

4. Biosafety

5. 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.

6. 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.

7. 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.

8. Staining

9. 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

10. 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

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

12. Heat-Fixing a Bacterial Smear

13. Gram Stain (primary stain) (mordant) (decolorizer) (counterstain)

14. Examples of Gram-Positive and Gram-Negative Organisms
Staphylococcus xylosus
Bacillus megaterium
Escherichia coli

15. (decolorizer/counterstain)
Capsule Stain
NO HEAT FIXATION!
(primary stain)
(decolorizer/counterstain)
Alcaligenes faecalis
Enterobacter aerogenes
Klebsiella pneumoniae
Serratia marcescens

16. Acid-Fast Stain Mycobacterium smegmatis (primary stain) (decolorizer)
(counterstain)
Mycobacterium smegmatis
Figure 4.18

17. Endospore Stain Bacillus megaterium (primary stain) (decolorizer)
(counterstain)
Bacillus megaterium

18. Four-Way Plate Streak

19. Four-Way Plate Streak

20. Plate Counts

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

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

23. Media Types

24. Nutrient Agar (NA) Special features: Used for: None
Culture of non-fastidious organisms

25. Sabouraud Agar/Broth Selective media Special features: Used for:
Acidic pH (5.5)
Used for:
Culture of fungi (yeasts and molds)

26. Brain Heart Infusion (BHI) Agar
Special features:
None
Used for:
BHI agar deeps used to determine oxygen classification

27. 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

28. 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

29. Blood Agar Differential Enriched Special features: Used for:
Sheep’s blood (nutrients, hemolysis substrate)
Used for:
Culture of fastidious organisms
Distinguishing between bacteria by hemolysis patterns (esp. Streptococcus)

30. Chocolate Agar Enriched Special features: Used for:
Boiled sheep’s blood (nutrients)
Used for:
Growth of fastidious organisms (esp. Neisseria spp.)
Distinguish Neisseria spp. using oxidase test

31. 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.)

32. 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. faecalis
E. aerogenes
S. xylosus
E. coli

33. MacConkey Agar Selective Differential Special features: Used for:
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. typhimurium
S. epidermis

34. 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
E. coli
P. aeruginosa
S. xylosus
E. aerogenes

35. Snyder Test Agar Selective Special features: Used for: 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

36. Temperature Classification

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

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

39. Examples of Temperature Classifications
Characteristics
Examples from Lab
Psychrophile
Growth Range:  -5 to 20 C
Optimum Temperature: ~10 C
Pseudomonas fluorescens*
(*Actually a psychrotroph)
Mesophile
Growth Range: 20 to 45 C
Optimum Temperature: ~37 C
Escherichia coli, Micrococcus luteus, Serratia marcescens, Saccharomyces cerevisiae
Thermophile
Growth Range: 35 to 80 C
Optimum Temperature:  ~55 C
Bacillus stearothermophilus
Hyperthermophile
Growth Range: Above 80 C
Optimum Temperature: variable
Archea such as Pyrolobus

40. Oxygen Classification

41. BHI Agar Obligate aerobe Microaerophile Facultative anaerobe
Aerotolerant anaerobe
Obligate anaerobe

42. Sodium Thioglycollate Broth
Obligate anaerobe
Obligate aerobe
Aerotolerant anaerobe
Facultative anaerobe

43. Control with Chemicals and Antibiotics

44. The Disk Diffusion Assay Measures Sensitivity
Figure 10.9

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