1. Revision

2. Bacteriology 1
Lab 1

3. Staphylococci
Gram + Cocci
Cluster in arrangement

4. Gram stain

5. mannitol salt agar MSA selective and differential growth medium
high concentration (about 7.5%-10%) of salt
contain carbohydrate mannitol
Ph indicator : phenol red
Staphylococcus aureus produces yellow colonies
S.epidermidis
S. aureus

6. Catalase test Procedure:- 1- add few drops of H2O2 on a slide
Is used to differentiate between staphylocci & streptococcci
Staphylococci give positive result
Use 3% H2O2 (hydrogen peroxide )as substrate
gas bubble is produced for positive results
Procedure:-
1- add few drops of H2O2 on a slide
2- transfer a small amount of colony growth to the slide
3- observe the bubbling reaction

7. Coagulase test
Is used to differentiae between S.aureus and other staphylocci species.
S.aureus gives positive reaction
Fibrinogen (plasma) is the substrate
Fibrin (visible clot) is produced

8. DNase test
Is used to differentiae between S.aureus and other staphylocci species.
DNA is the substrate
The hydrolysis of DNA is detected by observing a zone of clearance
Procedure:-
1- Using a sterile loop, inoculate organisms on Dnase plate. Make sure each test area is labelled clearly.
2- Incubate the plate aerobically at 35oC for 13 to 24 hours.
3- Flood the surface of the plate with 1 N hydrochloric acid (HCL )solution. Tip off the excess acid.
4- Look for clearing around the colonies within 5 minutes of adding the acid.
zone of clearance

9. Summary + - Tests S.aureus Other Staphylococci Catalase test
Coagulase test -
Dnase test

10. Lab 2+3
streptococci

11. Streptococci
Gram + cocci
Chains in arrangement
Catalase negative

13. Types of hemolysis : Blood agar

14. Alpha hemolysis Partial hemolysis of RBCs
Mainly S.pneumoniae & S. viridans
To differentiate: optochin disc
S.pneumoniae is sensitive while S. viridans is resistant.

15. Beta- hemolysis Complete Hemolysis of RBCs
Mainly GAS (Group A streptococci ) & GBS (Group B streptococci )
To differentiate: bacitracin test
GAS is sensitive while GBS is resistant.

16. Gamma – hemolysis No hemolysis
Mainly Enterococci : E. faecium & E. feacalis
Can grow at 6.5 % salt NACL
Can hydrolyze esculin in 40% bile ( bile esculin)
Bile Esculin agar
- Bile is the selective agent
- Esculin is used to differentiate Enterococci from other bacteria
BILE inhibits gram + bacteria except enterococci & group D streptococci

17. AFB
Lab 5

18. Gram stain Gives positive or negative reaction
It does not applied to all bacteria
ex. Acid Fast Bacilli

19. Acid Fast Bacilli (AFB)
Why Gram stain is not used ?
- AFB cell wall has ~ 60% lipid (waxy). So, it makes gram stains impermeable.
Ex. Mycobacteria spp.
Mycobacterium tuberculosis (MTB)

20. AFB stain Two procedures : 1- Light microscope
- Ziehl-Neelsen and Kinyoun methods
2- Fluorescent microscope
- Fluorochrome method

21. Ziehl-Neelsen stain Procedure :-
1- Primary stain: carbol fuchsin , 5 minutes( basic fuchsin +phenol)
2- decolorizer : acid alcohol , 1-2 minutes
3- counterstain: methylene blue, 1 minutes

24. Ziehl-Neelsen (ZN)staining
Hot method by using heat
Cold method ( no heat ) is called Kinyoun
- increasing concentration of basic fuchsin and phenol

25. Summary Hot ZN staining Procedure :-
1- Primary stain: carbol fuchsin (basic fuchsin +phenol) , 5 minutes
2- decolorizer : acid alcohol , 1-2 minutes
3- counterstain: methylene blue, 1 minutes

26. Neisseria
Lab 6

27. Neisseria
Gram negative cocci
- diplococci (bean shaped)

28. Biochemical reaction
Catalase +ve
Oxidase +ve

29. Oxidase test
The Oxidase Test is used to identify bacteria containing the respiratory enzyme cytochrome c oxidase
TMPD

30. culture Chocolate medium + 5% CO2 (capnophilic)
Incubation: At 37ºC for hrs.
Colonial morphology: small, gray, translucent and raised.

31. Neisseria spp. : Carbohydrate Utilization test
only one carbohydrate source in the medium
Phenol red as pH indicator
Differentiate between N.gonorrhea & N.menengitidies

32. Neisseria spp. : Carbohydrate Utilization test
N. Gonorrhea
N. Meningitides
Glucose
Maltose
Lactose
N. Gonorrhea + -
N. Meningitides

33. Bacillus
Lab 8

34. bacillus
Gram positive Rod
Spore forming

35. Bacillus subtilis Culture :
- nutrient agar : creamy irregular colonies
- Blood agar : Beta-hemolysis

36. Endospore staining

37. Bacterial Endospores
Endospores are a dormant stage of some bacterium that allows it to survive conditions that would normally kill bacteria such as extreme drought or heat
Endospores ultimately function are protection for the bacterial genome
Endospores provide resistance against:
Low nutrient conditions
Radiation
High temperatures and various chemical disinfectants

38. The Vegetative Cell Gives Rise to One Spore
Bacterial Cell
Spore
Bacterial Cell
The endospore is able to survive for long periods
of time until environmental conditions again become favorable for growth.
The endospore then germinates, producing a single vegetative bacterium.

39. Not all bacterial species can form spores
A few genera of bacteria produce Endospore such as Clostridium and Bacillus
(Endospore production is associated with Gram Positive bacteria)

41. Schaeffer-Fulton Stain Procedure
1. Prepare a smear. Air Dry. Heat fix
2. Put the slide on steam rack
3. Flood the smear with Malachite Green stain
4. Steam slide for 5 minutes (be sure that the satin is not dry, if so , add more satin )
5. Drain slide and rinse with DI water.
7. Flood smear with Safranin (counter stain). This stains the vegetative cell. (Leave for 1 minute)
8. Drain the slide and rinse with DI water
9. Dry
10. View slide under 100x oil immersion.

42. Endospore Stain Example Spores: Green Cell: Red or Pink
Practical: Each student will make smear of Bacillus subtilis