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assalamualaikum w.b.

I attached down here a quick revision notes regards the microbiology


practical exam....

I know it's quite late but I hope with the time remains,it could help to
ease our revision before the examination. .

this note DOESN'T include the mycobacteria, serological techniques &


antibiotic sensitivity test...so,u need to revise these subjects by your
ownself..

Any mistakes of mine,i'm really sorry...and i'm open for discussion..

these note is to ease ur revisions..feel free to add for urself any points
that u feel important..

thank u

-AFIQ FAHIMY-
2nd year
Section 3

Afiq Fahimy -Section 3


Page 1
STAPHYLOCOCCI

CLASSIFICATION (3 main species)

Coagulase +ve Coagulase -ve

-Staph.Epidermitis
Staph.Aureus
-Staph.saprophyticus

Morphology : Biochemical raction :

-Gram +ve cocci (violet) -Catalase +ve

-cluster (grape-like shape) (differentiation with streptococci)

-facultative anaerobes -Oxidase –ve

Normal flora : skin & mucous membranes

Afiq Fahimy -Section 3


Page 2
STAPHYLOCOCCI IN CULTURE MEDIA

NUTRIENT AGAR :

-Big pigments “needle-head pigment”

-vary in colours from golden yellow,lemon yellow or white

BLOOD AGAR :

-β-haemolysis

(big pigment,transparent differ than


streptococci which are minute pigments)

Afiq Fahimy -Section 3


Page 3
MANNITOL SALT AGAR (SELECTIVE MEDIA):

Yellow haloes : ferment mannitol salt agar à staphylococci

Pink haloes : non-ferment mannitol salt agars

Afiq Fahimy -Section 3


Page 4
STAPHYLOCOCCI AUREUS
ANTIGENIC STRUCTURE (STRUCTURAL VIRULENCE FACTORS):

(i) Peptidoglycan Polymer


(ii) Teichoic acids – adherence of organism
(iii) Protein A –antiphagocytic
-leading to coagglutination

(iv)Clumping factor

(v)Capsular polysaccharide

(vi)Surface receptor

PRODUCTION OF EXTRACELLULAR FACTORS

TOXINS ENZYMES

• Haemolysins • Coagulase : coagulates plasma

• Leucocidins • Staphylokinase :-fibrinolysis

-septic metastasis

• Enterotoxin(A-F) :

-causes of food poisoning • Hyaluronidase : spreading factor

• Exfoliative Toxin : desquamation of skin • β-lactamase : inactivates penicilin


-“Skin Syndrome” (no pus)

• Toxic shock syndrome Toxin-1


(TSST-1)

-superantigens

-causing Toxic Shock syndrome

Afiq Fahimy -Section 3


Page 5
CLINICAL FINDINGS

PYOGENIC INFECTIONS TOXIN MEDIATED ILLNESS

• Focal suppuration • Food Poisoning : Enterotoxin

• Toxic shock syndrome : TSST 1

• Dissemination with visceral


localization & suppuration
• Scalded skin syndrome : Exfoliative Toxin

SPECIAL CHARACTERISTICS OF S.AUREUS


• Coagulase +ve (differentiation with s.epidermitis & s.saprophyticus)

• Form yellow haloes with mannitol salt agar

• Liquefies Gelatin

Afiq Fahimy -Section 3


Page 6
Coagulase –ve staphylococci

Staph.Epidemidis Staph.Saprophyticus

DIFFERENTIATION
Sensitive Resistant
(Novo-Biocin test)

Disease : Disease :

- Peritonitis ( patients with peritoneal dialysis) Urinary tract infection in women of


- Chronic septicemia child-bearing age
- Endocarditis (heart surgery)
- Bacteraemic infections (indwelling catheter)

*have large amounts of


polysaccharide slime

Afiq Fahimy -Section 3


Page 7
STREPTOCOCCI

Haemolytic Serological Biochemical


Properties grouping properties

(A-U except I&J)

Important group

Differentiation
GROUP A GROUP B

(Strept.Pyogenes) (Strept.Agalactiae)

Bacitracin test

α- haemolytic:

-partial destruction of RBC

-green pigmentation

-Strept.Viridans

-Strept pneumoniae

Haemolytic
Properties
β- haemolytic:

-complete lysis of RBC

-clear zone around colonies

-Strept.Pyogenes
Non-heamolytic:

-No haemolysis

-No effect on blood agar

Afiq Fahimy -Section 3


Page 8
α- haemolytic:

-partial destruction of RBC

-light does not clearly


pass through

-green pigmentation

-Strept.Viridans

-Strept.pneumoniae

β- haemolytic:

-complete lysis of RBC

-clear transparent zone of


haemolysis

-Strept.Pyogenes

Afiq Fahimy -Section 3


Page 9
SLIDE IDENTIFICATION

Streptococci in chain

e.g :streptococci Pyogenes

Gram +ve (violet)

Long chains cocci

Gram +ve Diplococci

e.g :streptococci pneumoniae

minute spherical in pairs and


sometimes form small group

differ to diphteria slide is to:

(i) the diplococci can been seen clear


it’s spherical shape
(ii) the shape of diptheria is rod-like
shape.

*both picture are pneumoniae

Afiq Fahimy -Section 3


Page 10
Streptococcus Pyogenes
Morphology : -gram +ve cocci (chains)

-facultative anaerobes

Normal flora : throat & nasal cavity

Clinical findings:

• Inflammation & suppurative conndition


• Streptococcal Toxic shock syndrome : Exotoxins A or B
• Post-streptococcal Infection : Glomerulonephritis
Rheumatic Fever

Biochemical reaction :

- catalase –ve

-sensitive to bactracin (differentiation with other group of β-haemolytic )

Afiq Fahimy -Section 3


Page 11
ANTIGENIC STRUCTURE TOXINS AND ENZYMES

• Capsule • Streptokinase : fibrinolysins

• Streptodornase :depolymerizes DNA


• Cell wall
(i) Group specific carbohydrate (C-Ag)

-serologic grouping (Lancefield) • Hyaluronidase : splits hyaluronic acid

-Serogrouping test of β-haemolytic strept are


• Streptococcal Pyogenic Exotoxins(SPE)-
done by precipitation technique using A,B,C

appropiate antisera Type A : Scarlet fever

Type A & C : encode for bacteriophage


(ii)Proteins (M,T,R)
also causing:
Important : Protein M
Streptococcal toxic shock syndrome
(inhibit phagocytosis)
Necrotizing fascilitis

• Haemolysins:

(a)Streptolysin O (SLO)

-oxygen labile

-diagnosis of rheaumatic heart fever

(b)Streptolysin S(SLS)

-not antigenic

-Oxygen stable

Serologic test : Antistreptolysin O titre

(ASO)

(more than 200 = rheumatic fever)

Afiq Fahimy -Section 3


Page 12
STREPT.VIRIDANS & STREPT.PNEUMONIAE
Characters Strep.Pneumoniae Strep.Viridans

Morphology Ovoid or lancelated Short or long chains of


diplococci rounded cocci

Normal flora Nasopharnynx Upper respiratory tract

Capsule Present Absent


Optochin test Sensitive Resistant
Bile solubility +ve (clear solution) -ve (turbid)
Inulin fermentation +ve -ve
Virulence in mice Pathogenic Non-pathogenic

Clinical findings Broncho-pneumaniae Subacute bacterial


(common disease) (remains harmless unless it is endocarditis (SABE)
provoked by viral infection)

STREP.VIRIDANS

STREP.PNEUMONIAE

Afiq Fahimy -Section 3


Page 13
STREP.PNEUMONIAE

VIRULENCE FACTOR :

(i)Pneumolysin O : lyse RBC under anaerobic condition

(ii)Neuraminidase : promotes pneumococcal access to the lung

(iii)Ig A proteases : cleave secretory Ig A

TYPING OF STREP.PNEUMONIAE

CAPSULLAR SWELLING REACTION (Quellung Reaction)

-interaction between the capsular polysaccharide and it’s corresponding


Ab(antiserum)

-useful for rapid identification and typing eihter in sputum or culture

STREPTOCOCCI PNEUMONIAE

Afiq Fahimy -Section 3


Page 14
NEISSERIA

Neisseria smear by Gram stain

Neisseria smear by
methylene blue stain

Differentiaton from the TB


slide is by:

(i)the neisseria is also stain


blue against the blue
background

(ii)in TB,the TB is stain red


against the blue background

Afiq Fahimy -Section 3


Page 15
NEISSERIA MENINGITIDIS
Morphology :

-Gram –ve diplococci

-aerobe organism

Normal Flora : nasopharynx

Culture:

Enriched media :Blood agar & Chocholate blood agar

Selective Media : Thayer Martin media

Biochemical reactions :
-Oxidase +ve

-Acid production from glucose and maltose

(differentiation of N.meningitidis with other neisseria)

Determinants of pathogeneticity :

(i)capsular polysaccharide

(ii)Lipopolysaccharide (LPS) – endotoxin for N.Meningitidis

(iii)Pili

(iv)Ig A proteases

(v)Outer membrane protein

Clinical Findings:

Meningeococaemia --> waterhouse Frederichsen syndrome

-->Meningitis (common complication)

Treatment :

Penicilin G

Chloramphenicol (for hypersensitivity patients)

3rd Generation of Cephalosporins

Carrier : rifampicin & Ciprofloxacin

Afiq Fahimy -Section 3


Page 16
CORYNAEBACTERIA

Look for the rod shape

to differentiate it with
diplococci pneumoniae
which are spherical..

Morphology :

-Gram +ve bacilli

-“chinese letter writings”

-show clubbing at both ends

-Contain metachromatic granules (Neisser method to see it clear)

-aerobes and facultative anaerobes

Biochemical reactions :

-Catalase & nitrate +ve

-Oxidase & Urease –ve

-Ferments glucose & maltose with acid production

Normal Flora : Respiratory tract,vagina,anterior urethra,skin

Afiq Fahimy -Section 3


Page 17
Culture:

-Enriched media :Loeffler’s serum (apppear white colonies)

-Selective Media : Mcleod’s Tullerite blood agar (appear grey or black colonies)

-Blood agar

Afiq Fahimy -Section 3


Page 18
DIPHTERIA TOXIN

• powerful exotoxin
• Only lysogenised strain are toxigenic & virulent
• heat labile
• Highly toxic
• Highly Anitgenic
• Separate into 2 fragments :

Fragment A : inhibit polypeptide chain elongation in the presence of NAD by inactivating the

elongation factor-2 (EF-2)

Fragment B : transport for fragment A

Toxigenicity(virulence) tests

• Elek’s test
• PCR
• Enzyme-linked immunosorbent assays (ELISA)
• Historical in-vivo test

Treatment :

• Diphteria antitoxic serum (I.M or I.V)


• Antibiotics : are given with antiserum

Prophylaxis :

• Fluid toxoid
• Aluminium precipitated toxoid (common combined with tetanus toxoid(T) and/or with pertussis
Vacccine(P))

(i)DPT (below 6 years of age)

(ii)DT (after 6 years of age)

Afiq Fahimy -Section 3


Page 19
EMPTY MEDIA
BASAL MEDIA

Fluid Media

NAME OF MEDIA CHARACTERISTICS


Peptone water

Constituents: peptone,NaCl & Distlled water

Sterilization : Autoclave (110°C for 10 min)

Uses : base for all sugar media

Nutrient Broth

Slightly light yellowish than the peptone water


as it contain meat extract

Constituents : peptone water , NaCl and meat


extract

Sterilization : Autoclave (110°C for 10 min)

Uses : base for most media for growth of


microorganisms

Afiq Fahimy -Section 3


Page 20
SOLID MEDIA
NAME OF MEDIA CHARACTERISTICS
(in tube)
Soft Agar In small test tubes

Constituents: Broth + 0.7 % agar

Sterilization : Autoclave (120°C for 20


min) but the medium is distributed in
small narrow tubes.

Uses :
Test motility of bacteria

(in tube)
• Deep Agar
Constituents : Broth + 2-3% Agar

Sterilization : Autoclave (120°C for 20 min)

Nutrient agar slope :

Uses :
to differentiate bacteria’s growth according
oxygen requirement

(in plate) Constituents : Broth +2 % agar


Nutrient Agar plate

Sterilization : Autoclave (120°C for 20 min)

Uses:
1)Keeping stock for cultures for stab
inoculation

Afiq Fahimy -Section 3


Page 21
ENRICHED MEDIA

SOLID MEDIA
NAME OF MEDIA CHARACTERISTICS
Blood Agar

Uses :

1)Growing delicate or fastidious


organisms

2)As a differential medium according


their haemolytic action

Chocolate Blood Agar

Uses :

1)Culture of Haemophilus &


Neisseria

Afiq Fahimy -Section 3


Page 22
NAME OF MEDIA CHARACTERISTICS
Loeffler’s Serum

Uses :

demonstrate morphology of
C.Diphteria

Egg Saline Medium

Uses :

Culture of Tubercle Bacilli

Afiq Fahimy -Section 3


Page 23
SELECTIVE MEDIA
NAME OF MEDIA CHARACTERISTICS
MacConkey’s Medium
Constituents : peptone water,agar, bile
salt,lactose,neutral red)

Selecting agents/inhibitory substance :


Sodium taurochlorate – inhibit
growth of gram +ve

Sugar : Lactose
(fermented by prevalent intestinal
organism,e.g: E-coli)

Indicator : Neutral red(to give


differential colour)

Sterilization : Steaming (30-60 min)

Uses :
To grow Gram –ve bacilli

Thiosulfate Citrate Bile salt Agar (TCBS)


Constituents :
Basal medium,Na thioslphate,Na citrate,
Bile,sucrose,Bromothymol-blue

Indicator : Bromothymol-blue

pH : 8.0 -8.6

Sterilization : steaming (30-60 min)

Uses :
Selective isolation of Vibrio
Cholerae

Afiq Fahimy -Section 3


Page 24
NAME OF MEDIA CHARACTERISTICS
Mannitol Salt Agar

Selecting agent : NaCl (10-15%)


(allows growth of staphylococci and
inhibits other organisms)

Sugar : Mannitol

Indicator : Phenol red

Uses :

Growth for staphylococci

Lowenstein Jensen Medium (L.J)


Constituents :
beaten eggs,mineral salts,malachite
green

Selective agent : Malachite green


(inhibit growth of bacteria except TB)

LJ + Glycerol :
Enhance growth of human type
(M.Tuberculosis)

LJ + pyruvate :
Enhance growth of bovine type
(M.Bovis)

Uses :
cultivation of tubercle Bacilli

Afiq Fahimy -Section 3


Page 25
ENRICHMENT MEDIA
NAME OF MEDIA CHARACTERISTICS
Selenite F-Broth

A liquid media

Not favouring the growth og E-coli

Uses :
Favour growth of
Thypoid & Parathypoid

Afiq Fahimy -Section 3


Page 26
SUGAR MEDIA
Sugar Media
No fermentation on sugar (phenol indicator-red)

Contain inverted Durham’s


tubes to trap air bubbles in case of
Fermentation with acid production fermentation with gas
-phenol change colour to yellow(acidic) BUT
with NO air buble trapped in Durham’s tube
Constituents:
Peptone water + 0.5 – 1.0 % of sugar to
be tested

Indicator : Phenol red or Andrade

Fermentation with acid and gas


production
-Phenol change to yellow & air bubles are
trapped in the durham’s tubes

Afiq Fahimy -Section 3


Page 27
Heat Resistant test :

Enterococci

Important species : Enterococci faecalis

Normal flora : Lower intestinal tract & faeces

Culture :

-ordinary culture media

-macConkey’s agar – minute lactose fermenting colonies

-blood agar –various degree of haemolysis & sometimes non-haemolytic

-media with high salt content ,NaCl(6.5%)

Selective test :

Heat resistance test : withstand heat at 60°C for 30 minutes

(still grow on all of the 4 quadrants)

Clinical Findings:

Urinary tract infections, endocariditis , biliary tract infections , ear infections, suppurative abdominal
lesions

Afiq Fahimy -Section 3


Page 28
OPTOCHIN,BACITRACIN & NOVOBIOCIN (How to know them?)

Novobiocin Test

They are test on the nutrient agar medium.

(it’s easy to identify)

Optochin Test

They are test on the blood agar medium.

The line of resistant are partially


haemolyse differ than the bacitracin which
are complete transparent haemolysis

Bacitracin Test

They are test on the blood agar medium.

The line of resistant are complete


transparent haemolysis

Afiq Fahimy -Section 3


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OTHER PICTURES :

Phage typing à tracing source of infection

àepidemiological study

Antibiotic sensitivity test

Afiq Fahimy -Section 3


Page 30
Single Radial Immunodiffusion

Widal Rack

Afiq Fahimy -Section 3


Page 31
-ve Serum

(haemolysis)
+ve Serum

(no-haemolysis)

Complement Fixation Test (C.F.T)

Afiq Fahimy -Section 3


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ELISA

Afiq Fahimy -Section 3


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