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Sterilization
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.
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 Nesisseria
meningitides &haemophilus
sterilize by : autoclave
type: enriched media
Sterilize by: heating in inspissator (oven) at
80c for 2h for 3 successive days to kill
bacterial spores

Fig. 2.8: TCBS media
-Green, Transparent medium
- It is used for isolation of Vibrio cholera
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

Used for: cultivation of anerobic
bact.
Suitable for growth of clostridia &
other anaerobes.
sterilize by : autoclave
Used for: cultivation of anaerobic bact.
Indicator: methylene blue

Bacterial Identification
Fig. 3.1: Gram-positive bacilli
(violet in colour)
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 الامتحان مرقمه ومين اكتر دواء يستخدم كعلاج
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: Pneumococci
in tissue, gram stain
gram positive capsulated
diplococci

Blood culture
Added to 50 – 100 ml fluid medium
5 – 10 ml blood
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.

3. Toxin-Antitoxin Neutralization Test
Antistreptolysin test O (ASO)
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
1/25 1/50 1/100 1/200 1/400 1/800 control
Titer 1/400
Fig. 2. 11: Antistreptolysin O Titer
(ASO)

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)
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 positve
bacteria +nystatin for fungi +cholesyin for gm
negative bacteria )
• Antibiotics are put because it is separated
from vagina or urethera.

CORYNEBACTERIUM GROUP
Fig. 4.3: Coryn.
Diphtheria in culture
methylen blue stain
Fig. 4.4: Coryn. Diphtheria
(Gram positive bacilli
have Chinese letter
arrangement)
Media: loffler’s serum agar.
Stain: gram & methylene blue
Test: Elek’s test

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
Positive test
Fig. 4.6: Elek’s test
Test strain
Precipitation band
Negative test
Artiserum
in the strip.

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
(pseudomonas & Vibrio
are ) oxidase psoitve
Fig. 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
Spirochaetes
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 antiantibody
with fluorescent die. To
identify treponema pallidum

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
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
Mention type of test to identify the candida organism : Germ tube
Mention 2 type for commensals of candida: vagina & alimentary tract &
mouth

VIROLOGY
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

immunology
E. Immunofluorecence
Fig. E-1: Direct immunofluorecence
for detection of specific antigen
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.

F. Enzyme Linked Immunosorbent Assay (ELISA)
-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.
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

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