System of bacterial identification
(Laboratory diagnosis)
Microscpe
: shape, arrangement , staining reaction(Gram or Z.N.)
motility
spore
(fix bacterial smear from colonies)
Culture (Blood agar) : rapidity of growth colony morphology
O2 or CO2 requirement
Temp. requirement for growth
Biochemical tests : production of acid from carbo. Fermentation
presence of certain enzymes
production of end metabolic product
Serological typing :
detection of antigen or
,,
antibodies
Analysis of metabolic product :
Genetic analysis :
by chromatography
by DNA probe (by PCR)
Staphylococci
M.O.
Disease
specimen for bacteriology
S.aureus: mastitis (cattle),
turbid or bloody milk
dermatitis, abscess (rabbit)
Pus or pus swab
Pus of spermatic cord (botryomycosis)
,
suppurative infection (man)
urinary, uterine, after operation
food poisoning (man),
Bumble foot (poultry)
food or vomits
inflamed foot
S.intermedius : pyoderma in dog
Pus or pus swab
S.hyicus
: primary pathogen of pig
S.epidermidis: normal flora, not cause disease but can invade
traumatized skin
S.saprophyticus: normal flora
Microscopic Exam.: grape like , Gr+ve cocci from infected tissue
(violet grapes) or Fix smear from colony after culture on a medium
1
Staphylococci
Stained colony from nutrient agar medium
Stained in tissue
Culture
Pus ( closed abscess)
Hemolysis
(pathogenic)
Blood agar(37C/24h.)
(open abscess ,milk sample ,
exudates from dermatitis
No hemolysis
Non pathogenic
mannitol salt agar
(37C/ 1-7days)
Yellow surronding
Colonies
red surronding
Colonies
(+ve mannitol )
(-ve mannitol )
Acid prod.
No acid prod.
chicken suspected food poisoning
Paired parker
medium
black colony
(+ve tellurit
Gram stain of smear from
colony record morphology
reduction
No black colony
(-ve tellurit
reduction
Hemolysins
4) Hemolysins are exotoxins that are membranedisrupting. They lyse host cells by creating
pores in their membrane.
5) Called hemolysins because erythrocytes are a
convenient cell type for assaying activity.
However, these toxins are usually toxic for
many cell types.
6) So hemolysin of Staphylococci disrupts RBCs
thereby increasing free iron but also causes
lysosomal disruption in neutrophils and is
cytocidal for other cells. In addition, it can
cause dermonecrosis and vasoconstriction.
Staphylococcus aureus
showing yellow pigmented colonies
surrounded by zone of Beta- haemolysis
Beta haemolysis
Yellow colonies
2
Staphylococcus epidermidis
White non –hemolytic colonies on •
blood agar
White
colonies
3
Mannitol salt agar
This is a selective media •
contains 7.5% NaCl –
***Staphylococci are one of the few •
organisms that grow in the presence of this
high a salt concentration****
Media also contains mannitol (sugar) and •
a pH indicator that turns YELLOW if
manitol is fermented
S. aureus ferments mannitol (Mannitol +Ve) •
other Staph. spp. grow on media, but don’t •
ferment
Mannitol Salt agar
Selective medium for Staph.aureus , yellow colour
indicate mannitol fermentation
Growth AND Fermentation
Growth but NO fermentation
Milk agar
Used to enhance pigment production
S.aureus produce golden yellow pigment
S.epidermidis produce white colonies
Paired Parker Medium
Staphylococcus aureus reduce tellurite producing black
colour and produce Lipase forming halo clear zone.
Enzymes (Exotoxins) for dignosis (pathogenic markers)
1-Coagulase(bound and free types)
2-Thermonucleases
Turbidity
Other enzymes (exotoxins):
1-DNAase : culture medium + DNA
Correlate with coagulases
Incubate at
+ HCL
37C/24h.
2-Hemolysins: alpha,beta,gamma (all pathogenic m.o. are
hemolytic)
3-Phosphatase: Culture medium + phosphate
m.o. produce
ammonia + indicator
pink color
4-Enterotoxins : m.o. grow in food (resist boiling for 0.5 h)
emetic centers
vomiting
5-Penicillinase : culture medium + penicillin
incubate 37C/24h
if m.o.grew on medium
m.o. contain the enzyme
6- Epidermolytic toxin
dermonecrosis
7-leukocidin
kill polymophs and macrophages
Coagulase
Used to identify pathogenic staphylococci.
• Staph. epidermidis: Coagulase (-)
• Staph. aureus & Staph. intermedius:
Coagulase (+)
Two types of coagulase enzymes have been
identified…
•Bound coagulase: Converts fibrinogen to fibrin
directly with no involvement of plasma factors.
Slide test detects ONLY BOUND coagulase.
Coagulase (cont’d)
• Free coagulase:
Staphylothrombin
Staph.
Coagulase
+
Coagulase
reacting factor
(prothrombin)In
rabbit plasma
fibrinogen
fibrin
(soluble)
(clot)
Tube test: Detects both free AND bound
Coagulase test
We will use tube test (rabbit •
plasma). This detects both bound
AND free coagulase. Check at
24 hours.
Coagulase test: (tube test)-
4
Staphylococcus aureus
Coagulase test:1) Slide coagulase
2) Tube coagulase
Negative
Positive
Zagazing University
Fac. Of Vet. Med.
Dep. Of Microbiology
Negative
Positive
Staphylococcus aureus
Growth on mannitol salt agar
DNase activity
S.aureus inoculum
Red colour indicate
fermentation
Clear zone
5
DNase reaction
Pathogenic Staphylococcus (rose pink)
Non-pathogenic Staph. Stained blue.
Differentiation of staphylococci from other cocci
Staph.
Catalase +ve
OF(fermentative)
Bacitracin resistant
1-Micrococci :
Catalase positive
Oxidative reaction( OF test)
bacitracin sensitive
2-Streptococci :
Catalase negative
Fermentative reaction (OF test)
3-Corynebacterium equi : Catalase positive
OF(
unreactive)
Antibiotic Sensitivity Test (Diagnostic test)
Bacitracin Sensitivity test of Micrococci showing
wide inhibition zone( > 10 mm)
pigment production
There are 2 types of Micrococci , one producing
orange pigment and other produce white pigment.
Oxidation Fermentation test
(O F test)
Unreactiv
Unreactiv
anaerobic
aerobic
Paraffin
layer
Oxidative
Unchanged
Unchanged
Coagulase negative staphylococci
Staphylococcus
saprophyticus
Novobiocin sensitivity
S.epidermidis (sensitive)
Novobiocin disc
Zone of inhibition
White non hemolytic colonies
Zagazing University
Fac. Of Vet. Med.
Dep. Of Microbiology
S.saprophyticus (resistant)
6
Phage typing
Aim : Epidemiological purpuses
1-human cases of S.aureus food poisoning
2- Bovine mastitis of S.aureus
Materials : -Culture medium for unkown isolate
- make wells in culture medium
-Inoculate culture medium with the unknown
isolate
- Put typing phage (Known ) in the wells
- Incubate 37C/24h.
Reading results : +Ve (growth inhibition of m.o. around wells
-Ve ( No
,,
,,
,,
S.aureus bacteriophage 7
typing
Confluent lysis
by phage
Zagazing University
Fac. Of Vet. Med.
Dep. Of Microbiology
Case #1:
History :
•
Milk sample from a cow that recently calved. •
swollen quarter fever systemic illness –
abnormal milk –
Macroscopic: round, white colonies •
with double-zoned hemolysis
Microscopic: –
GRAM POSITIVE COCCI •
Catalase –
POSITIVE and so the next step was - •
Coagulase
POSITIVE –
Isolate was Staphylococcus aureus
Case #2 •
History : Milk sample from a clinically normal animal. •
“Normal cow” at dry-off •
Macroscopic: medium, round white –
colonies - no hemolysis
Microscopic –
Gram positive cocci •
Catalase –
POSITIVE so did a •
Coagulase –
NEGATIVE •
Isolate was Staphylococcus epidermidis
Case # 1 vs. Case # 2
Both isolates were staphylococci •
However, in Case # 1: S. aureus is •
considered to be a major mastitis
pathogen, with significant herd
implications.
With case #2: S. epidermidis is a •
normal inhabitant of the skin, and
was likely simply a contaminant
Review of Lab 4
Isolate 1: wound infection
Positive
Macroscopic: round, white, medium •
sized with double-zoned hemolysis
Gram positive cocci Microscopic: •
Catalase: •
Positive
Coagulase: •
Mannitol salt: •
Grew AND Fermented mannitol
Organism was S. aureus
Isolate #2: wound infection
Macroscopic: round, white, small sized •
with no hemolysis
Microscopic: •
Gram positive cocci
Catalase:
•
Positive
Coagulase: •
Mannitol
salt: •
Grew; did not
ferment mannitol
Negative
Isolate was S. epidermidis
Question for thought
In the case (the dog with the wound •
infection), what could you do to try
to differentiate INFECTION with
Staphylococcus spp. from
contamination?
Case #4 Dog: dermatitis
Macroscopic: white, medium, round •
with double-zoned hemolysis
Microscopic •
Gram positive cocci –
catalase •
positive –
puts the organism in the genus –
Staphylococcus
Organism was most likely S. intermedius
The most important diagnostic enzyme for staphyloccoci is.
a-haemolysin. b-coagulase. c-transferase. d-lipase.
They are specifically selective for staphyloccoci.
a-Baired parker meduim.
b-mannitol salt agar .
c-selective maconckey’s media.
d-a and b.
For phage typing of unknown m.o we need .
a-known phage. b-unkown phage (live). c-dead phage.
d-non of them.
Bulk amount of milk contains staphyloccoci .
a-condemn it.
b-detect if the m.o. is pathogenic .
c-turned to sterilization.
d-add antiseptic.
Bound coagulases in pathogenic staphyloccoci are detected through .
a-slide coagulase. b-tube coagulase. c-precipitation test. d-CFT.
Pus in the foot of a hen, what of them is suspected to be examined.
a-S.aureus. b-E.coli. c-salmonella.
d-anthraciod bacillus.
Micrococci are non pathogenic, to differentiated them from pathogenic
staphyloccoci , run for.
a-bacitracin sensitivity. b-optchin susceptibilty. c-gram staining.
d-bipolarity
Materials
)per group of 4(
BA culture of each of 4 isolates
1
BA plates inoculated from broths on Tuesday
bottle of 3% H2O2
1
)Mannitol salt agar plate (MS
1
)tubes of rabbit plasma (already diluted 1:5
2
blood agar plates
2
sterile plastic bulb pipettes
2