1. طريقة تخطيط الاطباق Streak Plate Method
التخطيط المتعامد

2. Streak Plate Method

3. Gram Stain 2. Saturate the smear with crystal violet for 1 minute.
spread as a thin layer
One drop of water +
Loop of Bacteria
1-allowed to air dry
smear
2-fixed
clean glass slide
2. Saturate the smear with crystal violet for 1 minute.
3. Rinse the slide gently with water.
4. Saturate the smear with iodine for 30 Second.
5. Rinse the slide gently with water.
6. Decolorize (alcohol)
7. Rinse the slide gently with water.
8. Saturate the smear with safranin for 1 minute.
9. Rinse the slide gently with water.
10. Carefully blot the slide dry with bibulous paper.
11. Observe the slide under the microscope

4. Gram staining Results Gram positive bacteria will stain purple.
Gram negative bacteria will stain red/pink.
Gram positive bacteria have a thick cell wall made of peptidoglycan,
whereas gram negative bacteria have thin layer of peptidoglycan.

5. Cells Description under the microscope
1- Cell response : GV + or GV-

6. Cells Description under the microscope
2- Cell shape and arrangement :
Cocci:
single- Diplo – Grape like-Strepto

7. Cells Description under the microscope
Road: Short or long
Single or Chain

8. Classification of Bacteria

9. Gram-Positive Bacteria

10. Gram-Positive Cocci

11. Catalase test
The catalase test is distinguished streptococci from staphylococci
flood culture with drops of 3% H2O2
Catalase-positive cultures bubble at once
The test should not be done on blood agar because blood itself will produce bubbles
H2O2
Catalase
H2O + O2 (gas, ↑)
Staphylococci

12. Species of Satphylococci
Three species of staphyloccoci have medical importance:
S. aureus: Pathogenic & commensally found in nose (nares)
S. epidermidis: non pathogenic & common commensals in nares & skin
S. saprophyticus: Cause UTI in female & occasionally commensally found skin

13. Coagulase Test Principle:
This test used to differentiate between S. aureus (CPS) & other Staphylococcus species (CNS)
Fibrinogen
(Plasma)
Coagulase
Fibrin
(Clot)

14. Staphylococci General characters: Gram Positive Cocci Grape-like
Non Motile
Non Spore Forming
Non Capsulated
Non Fastidious
Facultative Anaerobes
Fermentative
Catalase positive

15. Gram stain of Staphylococcus

16. Laboratory diagnosis of Staphylococcus
Gram Stain:
Gram Positive Cocci, arranged in cluster
Culture: 1- Blood agar , 2- MSA media
Blood agar (Non-Selective Media)
Coagulase Positive Staphylococci are Pigmented & hemolytic
Coagulase Negative Staphylococci are non-pigmented & non-hemolytic

17. MSA is selective differential medium for staphylococci
It contains: NaCl (7.5%), Mannitol, & Phenol Red
The cause of selectivity due to presence of high salt concentration
The cause of differential because contains mannitol (sugar) and phenol red (pH indicators turns yellow in acidic pH and turns red in alkaline pH).

18. Coagulase Test The tube coagulase test (Free): Procedure: Advantage
Mix 0.1 ml of culture ml of plasma
Incubate at 37C for 4 h
Observing the tube for clot formation
Any degree of clotting constitutes a positive test
Advantage
More accurate
Disadvantage
Time consumed
S. aureus
S. epidermidis

19. Coagulase Test Two Methods: The slide Method Tube Method
The slide coagulase test
Used to detect bound coagulase or clumping factor
Add one drop heavy bacterial suspension and one drop of plasma on clean slide
Mixing well and observing for clumping within 10 seconds
Advantage
Rapid diagnosis
Disadvantage
Less accurate

20. Deoxyribonuclease (DNAase) test
Principle:
DNA is insoluble in acid
DNA is hydrolyzed into oligo nucleotides by the action of DNase
Nucleotides soluble in acid

21. DNase Test Procedure & result:
Inoculate DNA agar with tested organism in circular motion
Incubate at 37C for 24-48h
Observe DNase activity by adding 1N HCl to the agar surface, a zone of clearing indicates a positive test
The zone represents the absence of DNA
The medium around colonies not producing DNase remains opaque, which is a reflection of the precipitation of DNA by the added acid.

22. Novobiocin Sensitivity
A simple disk diffusion test for estimating novobiocin susceptibility used to distinguish S. saprophyticus from other clinically species
Inoculated overnight culture on Mueller-Hinton agar
Add novobiocin disk on inoculated plate
Incubate at 370C overnight
Novobiocin resistance is intrinsic to S. saprophyticus but uncommon in other clinically important species.

23. Practical Work Gram stain Catalase test Mannitol fermentation on MSA
Coagulase Test by Tube and Slide Method
DNAase Test