Fig. 1.1: Simple laboratory

autoclave(moist heat) Temp. 121°C (due to double atmospheric pressure)

Time : required: 20 min.

Used for: surgical inst ,gauze,cotton,&culture media that

are destroyed by heat.

It is an efficient method of sterilization due to penetration

power of steam &high temp.

Sterilization

How to test efficacy of autoclave?

By 1. chemical method :by cultivation of bacillus sterothermophilus( that can survive up to 120 c and beyond that it die) then it is cultivated to test efficacy.

2. biological method :by chemical indicator on a tape that changes its colour into black or dark purple if the autoclave is efficient.

Fig. 1.2: Hot air oven(dry heat).

Temp. 160°C Time required: 1 hour

Used for: metal & glass – ware

equipment &powder and oil.

With discs have varying

diameter & varying pore size

• Suitable for large volume of

fluid.

• and any biological fluids

contain proteins that would

coagulate if sterilized by

autoclave as serum, vaccines

and media used for cultivation

of viruses

N.B.

Bacterial loop : ( enoculating needle )

Sterilized by red heat

PLASTIC SYRINGE:

By ethylene oxide gas or gamma rays.

Fig. 2.1: Blood agar opaque red in color

Type:

Enriched & indicator medium

Differentiate bet bact. By their hemolytic

action on red cells (complete , partial, no

hemolysis) .it is not sterilized by autoclave

The sterile blood is added to sterile agar at

temp. of 55 and poured in sterile plates

Fig. 2.2: Blood agar showing:

-Alpha heaemolysis (partial & greenish)

-Beta haemolysis (complete )

Media

Fig. 2.3: MacConkey’s medium

Reddish transparent medium

Fig. 2.4: MacConkey’s medium showing:

-Lactose fermenter → rose pink colonies

-Non Lactose fermenter → pale yellow colonies

Type: indicator (differential media)

indicator: neutral red

sugar content : lactose

Use: to differ. Between lactose , non lactose frementers and for isolation of enteric

media

Sterilized by autoclave

Suitable for: growth of Nesisseriameningitides &haemophilussterilize by : autoclavetype: enriched media

Sterilize by: heating in inspissator (oven) at

80c for 2h for 3 successive days to kill

bacterial spores

Growh after 6-8 weeks

Type: selective media

inhibitory substance by: malachite green that inhibit all bacterial flora exept

mycobacterium tuberculosis.

Sterilize by: heating in inspissator (oven) at 80c for2 hours for 3 successive days

Fig. 2.8: TCBS media

-Green, Transparent medium

- It is used for isolation of Vibrio cholera

Used for: cultivation of anerobicbact.Suitable for growth of clostridia & other anaerobes.sterilize by : autoclave

Used for: cultivation of anaerobic bact.Indicator: methylene blue

Fig. 3.1: Gram-positive bacilli

(violet in colour)

Bacterial Identification

Fig. 3.2: Gram – negative bacilli

(Red in colour)

Fig. 3.8: Catalase test

-Positive test shows

gas production

-Give positive: with

staphylococci

Fig. 3.11: Oxidase test

Positive test gives deep

purple color

Give positive: with

Neisseria , vibrio

&pseudomonas

Fig. 3.10: Urease test

Positve test gives Pink color

Give positive: with proteus

Indicator: phenol red

Fig. 3.14: Antibiotic sensitivity test

Use: To choose the most effective drug used for ttt.

Type: Disc diffusion method

The drug show the largest zone of inhibition is the most effective

هتيجى ف االمتحان مرقمه ومين اكتر دواء يستخدم كعالج

SENSITIVE (LARGEST ZONE OF INHIBITION)

Non sensitive (resistant)

Fig. 1.1: Staph.

Aureus on blood

agar it produce Beta

haemolysis

Fig. 1.2: Staph

aureus on nutrient

agar it produce

golden yellow

endopigment

Staphylococci

Systemic bacteriology

Or bunches or gape like

Fig. 1.6: Coagulase test Staph. Aureus gives positive coagulase

test

Fig. 1.5: Catalase test

all Staphylococci are

catalase positive

strepcoocci

Fig. 2.2: Growth of

Strept viridians on

blood agar showing

partial or alpha

heaemolysis

Fig. 2.1: Growth of

Strept pyogenes on

blood agar showing

complete or Beta

haemolysis

Fig. 2.3: Strept. In

culture gram stain.

(Gram positve cocci

arranged in chain)

Fig. 2.8: Optochin

sensitivity test

strept. Pneum is

sensitive

strept. Viridance is

resistant

Fig. 2.9: Bacitracin

sensitivty test

strept. Pyogens is

sensitive

strept. Agalactiae is

resistant

Fig. 2.4: Sterpt. In

pus Gram positive

cocci

pneumococci

Fig. 2.7:Pneumococci

, Quelling reaction

(capsule swelling with specific anti-sera)

Fig. 2.6: Pneumococciin tissue, gram stain gram positive capsulated diplococci

Added to 50 – 100 ml fluid medium

Use of Blood culture

for diagnosis of acute bacterial endocarditis

Disease diagnosed by bl. Culture:subacute bacterial endocarditis(streptococcus viridans) “ sanguis, salivarius” + post streptococcal disease , brucellosis ,typhoid fever and puerperal sepsis.

5 – 10 ml blood

Blood culture

Use:

1. For diagnosis of rheumatic

fever

2. detection of post

streptococcal infection.

3. Acute glomerulonephiritis

Type of test:

Neutralization (in vitro)

The presence of antitoxin in

sera neutralizes the hemolytic

effect of toxin on addition of

red blood cells.

Diagnostic test: >200 todd

unit

3. Toxin-Antitoxin Neutralization Test

Antistreptolysin test O (ASO)

Fig. 2. 11: Antistreptolysin O Titer

(ASO)

1/25 1/50 1/100 1/200 1/400 1/800 control

Titer 1/400

Neisseria menngitides

Fig. 3.2: Neisseria in

cluture Gram negative

cocci

Fig. 3.3: Pathogenic

neisseria in pus, gram

stain. (Intra &

exteracellular Gram

negative diplococci)

Fig. 3.4: Oxidase test

All neisseria is oxidase positive

Neisseria gonorrhea

• The media used for cultivation is Thayer martin media which is chocolate blood agar + antibiotics (vancomycin for gm positvebacteria +nystatin for fungi +cholesyin for gm negative bacteria )

• Antibiotics are put because it is separated from vagina or urethera.

CORYNEBACTERIUM GROUP

Media: loffler’s serum agar.

Stain: gram & methylene blue

Test: Elek’s test

Fig. 4.3: Coryn.

Diphtheria in culture

methylen blue stain

Fig. 4.4: Coryn. Diphtheria

(Gram positive bacilli

have Chinese letter

arrangement)

Fig. 4.6: Elek’s test Positive test

Test strain

Precipitation band

Negative test

Artiserum

in the strip.

Type of reaction: precipitation test (Ag- Ab reaction)

use: Detection of toxiogenic strain of C . Diphtheriae

it is a double immunodiffusion test diffusion of the organism toxin with the antitoxin forming precipitation band or line

Mycobacterium

Fig. 5.3: Myc. TB in Sputum,

Z.N stain

(few thin pink bacilli with blue

background)

Fig. 5.1: Selective

media

for Myc. T.B.

Fig. 5.2: Culture of Myc.

TB on L.J. media

- Grow after 6-8 week

Fig. 5.5: Tuberculin test It involves intradermal injection of Purified Protein Derivative (PPD)

type of test: Delayed type hypersenstivity used for Diagnosis of T.B.

Type of reaction: antigen-anibody reaction

+ve: give area of induration about 9 mm Time: 48 – 72 hours after injection

Media: lowensten jensen media stain: ziehl neelsen stain

Protaus

Fig. 8.4: Proteus culture

on Nut. Agar (swarming

growth)

Fig. 8.5: Proteus in

culture, gram stain

(gram negative bacilli

proteus showing

pleomorphism )

Fig. 8.6: Urease

test (proteus is

urease test passivit

+ve -ve

Fig. 8.9: Oxidase test(pseudomonas & Vibrio

are ) oxidase psoitveFig. 8.8:

Pseudomonas

culture on Nut.

Agar (produce

greenish blue

exopigment

Fig. 8.7:

Pseudomonas in

culture, gram stain(gram negative bacilli)

Pseudomanas

Spore forming gram-positive bacilli(Bacillus & clostridium group)

Fig. 9.4 Gram stained film of

clost. Tetani in culture -

Gram-positive long bacillus

with terminal plugging spore

(dram-stick) appearance

Fig. 9.6 Robertson’s cooked-meat broth medium glutathione is released after boiling (reducing agent)

Fig. 11.2 Fontana stained film showing commensally spirochetes

Type of stain:

Special stain

Fig. 11.3 Dark ground illumination microscopy of Treponema pallidum.

نادرا اما تنزل

Fig. 11.4:Indirect Immunofluorescence for spirochetes. It is called indirect as we put antibody then we put antiantibodywith fluorescent die. To identify treponema pallidum

Spirochaetes

Reading of the results:

- No hemolysis means a

positive reaction

i.e the complement is bound

to the antigen-antibody

complex.

- Hemolysis means a

negative reaction

Fig. C-2: Complement fixation test

wasserman test

wasserman test

MYCOLOGY

Mention type of test to identify the candida organism : Germ tube

Mention 2 type for commensals of candida: vagina & alimentary tract & mouth

Fig. 12.3 Germ tube test

for pathogenic strain of

C. albicans

Fig. 12.1 Gram’s

stained films of

Candida albicans

Fig. 12.2 Candida albicans culture on sabouraud’s dextrose agar

Fig. 1.4: CPE (cytopathic effect of Herpes virus)

Showing: Enlarged, aggregated and ballooned

cells causing multi- nucleated giant cells

Cell culture for vesicular lesion : herpes giant

cell virus

VIROLOGY

E. Immunofluorecence

Fig. E-1: Direct immunofluorecence

for detection of specific antigen

immunology

Direct immunofluorecence

- A specific antibody labeled with a fluorescent molecule (fluorescein or rhodamine) is added to the unknown antigen in the specimen.

- It is used to detect the presence of an antigen on a cell or tissue.

-It is used for detection of antigen, antibodies.-ELISA is based on the measurement of an enzymatic reaction associated with immune complexes as detected by color development.

F. Enzyme Linked Immunosorbent Assay (ELISA)

Fig. F-1: Plate of ELISA test

Immunologic reactions

1. Antistreptolysin o titer

2. Elek’s test

3. Direct immunoflourescence test

4. Elisa plate test

5. Tuberculin skin test