Microscope & safety rules

THE LIGHT MICROSCOPE

Has 4 systems
 Support system

 Optical (magnification) system

 Illumination system

 Adjustment system
 The microscope condenser is
responsible for
• Controlling the amount of light
• Magnifying the object
• Bringing the light into a strong beam
• Reducing the refractive index
Light Microscopes
 A biological safety cabinet (BSC)

• A biological safety cabinet

(BSC) is used as a primary
barrier against exposure to
infectious biological agents.
 A biological safety cabinet (BSC)

• A BSC has High Efficiency Particulate Air

(HEPA) filters.
• The airflow in a BSC is laminar, i.e. the air
moves with uniform velocity in one direction
along parallel flow lines.
Biological Safety Cabinets
Microbiology Laboratory tools
1- Bacteriological loop
Inoculating loop: a tool for transferring and
streaking cultures.
• Inoculating needles are used for preparing ‘stab’
cultures.
Hot plate with magnetic stirrer
• It is electrically powered equipment performs the
dual function of heating and agitation.
Identify & mention its uses?
Incubator
• To provid microorganisms with optimum
temperature for growth.
– temperature is maintained at 28-30°C for bacteria,
about 25°C for molds, and 35-37°C for mesophilic
bacteria.
Microbiology Laboratory tools

3. Petri dishes
Microbiology Laboratory tools

4. Test tubes
 Sterilization
&disinfection
Hot air

oven

It is used for dry heat

sterilization
 Dry heat:
Red heat
Incineration
Flaming
Hot air oven
• Advantages of dry heat:
• It is an effective method of sterilization of heat stable
articles.
• The articles remain dry after sterilization.
• This is the only method of sterilizing substances that are
damaged by moisture e.g. powders and oils.
• Disadvantages of dry heat:
• Since air is poor conductor of heat, hot air has poor
penetration.
• Glasses may become smoky.
• Takes longer time compared to autoclave.
What are the methods used in
moist heat sterilization?
 Examples of moist heat:
1. At temperature below 100°C:
a) Pasteurization.
b) Vaccine bath.
c) Inspissation.
2. At temperature 100°C:
a) Boiling water (100°C)
b) Steam at 100°C.
3. At temperature above 100°C: (Autoclave)
 Moist heat:

Moist heat at a temperature above 100°C (The

autoclave) :

Simple Single Drum

 (The autoclave)
• Benchtop / • Automatic
Sterilizer
AUTOCLAVE VERTICAL Steam-jacketed
autoclave
• Identify & mention its advantages?
 autoclave
Advantages:
– It has more penetrative power than dry air,
– It moistens the spores (moisture is essential for
coagulation of proteins),
– Condensation of steam on cooler surface releases
latent heat, condensation of steam draws in fresh
steam.
• Mention the ways of used to test the efficacy
of sterilization process?
• The efficacy of sterilization process is checked by:

– Physical method: Temperature chart recorder.

– Chemical method: Browne’s tube No.3 (its color changes from red to

green after 1h at 160 °C).

– Biological method: 106 spores of Bacillus subtilis on paper strips are

placed inside the hot air oven. After sterilization cycle, the strips are

removed and inoculated into thioglycollate broth or cooked meat

medium and incubated at 37°C for 3-5 days. Proper sterilization should

kill the spores.

• Classify disinfectants according to their mechanism
of action?
(a) Action on membrane (E.g., Alcohol, detergent)
(b) Denaturation of cellular proteins (E.g., Alcohol, Phenol)
(c) Oxidation of essential sulphydryl groups of enzymes (E.g., H2O2,
Halogens)
(d) Alkylation of amino-, carboxyl- and hydroxyl group (E.g.,
Formaldehyde)
(e) Damage to nucleic acids (Formaldehyde)
Staining
• Identify & mention other types of staining
techniques?
 simple staining
• Types of staining techniques:
• 1) Simple staining (Methylene blue)
• 2) Differential staining (gram stain, ZN stain)
• 3) Special staining (capsule, spore, flagella)
Simple Staining
(Methylene blue)
Methylene blue
stain
Steps of Gram
staining
Steps of gram staining
Steps of Z.N staining:
 Gram positive cocci

• Arranged in
clusters:
• Staphylococcus,
such as S. aureus
 Gram positive cocci
• Clusters: characteristic
of Staphylococcus spp.,
such as S. aureus

• Chain: characteristic
of Streptococcus spp.,
such as S. pneumoniae
, B group streptococci
 Staphylococcus Streptococcus
Arranged in clusters arranged in chains
Gram negative cocci
Gram positive bacilli
Gram negative bacilli
Violet stained gram-
positive cocci and pink
stained gram-negative
rod-shaped bacteria
Capsule appearance by gram
Background is
stained as well as
the bacteria and
there is a “halo”
around the
bacteria. The
halo represents
the capsule.
Ex. Klebsiella
pneumoniae.
Endspore staining by malachite green
Spore forming microorganism
 Spore
appearance by
gram stain
Carbol fuchsin
Flagella staining
Treponema by dark-field microscopy
Bacterial growth
Bacteria are divided into (according to their O2 requirement):
(a) obligate aerobes grow only at the surface.
(b) Anaerobes: grow only away from the surface.
(c) Facultative aerobes: grow in presence or absence of
oxygen but better growth occurs near the surface.
Bacteria are divided into (according to their O2 requirement):
(d) Microaerophiles requires small concentration of O2
(e) Aerotolerant anaerobes grow throughout the tube.
However, growth is no better near the surface.
CULTURE MEDIA
 (3) Types of media
• According to consistency:
• LIQUID media
• SOLID media.
• According to uses:
• Ordinary media: agar plate, agar deep, broth agar or agar slant
• Enriched media: they are enriched by addition of blood, serum or egg as:
– Blood agar: types of haemolysis on blood agar are Alpha, Beta or gamma.
– Chocolate Agar
– Loffler's Serum.
– DORSET EGG
• Differential media: they contain selective agents, enrichment and indicator as:
– MacConkey,s Media
– Lowenestien Jensen (LJ) media
– Thiosulphate-citrate-bile salt-sucrose (TCBS)
– Mannitol salt agar
• Anaerobic media.
• Transport media.
• Sugar media as Triple sugar iron media
Ordinary (basal) media
ENRICHED MEDIA
BLOOD AGAR
TYPES OF HEMOLYSIS ON BLOOD AGAR

• Alpha

• Beta
• gamma
 α-hemolysis
• The agar under the
colony is dark and
greenish.
• as Streptococcus
pneumoniae
 Beta hemolysis (β-hemolysis)
(complete hemolysis)
• It is a complete lysis of red cells in the
media around and under the colonies
• the area appears lightened (yellow)
and transparent.
• As Streptococcus pyogenes, or Group
A beta-hemolytic Strep (GAS) &
Staphylococcus aureus
B hemolysis on Blood agar
ex. S. aureus
 Gamma hemolysis (γ-hemolysis)
• Organism does not induce
hemolysis, the agar under
and around the colony is
unchanged.
• organism is called non-
hemolytic
• As Enterococcus faecalis
(formerly called "Group D
Strep") & staphylococcus
epidermidis
TYPES OF HEMOLYSIS ON BLOOD AGAR

-hemolysis

-hemolysis

-hemolysis
 ENRICHED MEDIA
Chocolate Agar
Composition: As blood agar 
but after addition of blood 
increase the temperature of 
water bath to 80 C for 10 min.
Uses: cultivation of Neisseria 
and Haemophilus
 Loffler's serum
(enriched media)
• culture medium containing horse
serum, meat infusion, and dextrose
for use in the cultivation
of Corynebacteria 
diphtheria
 
 MacConkey,s Media
(Selective and differential media)
It contains :

• bile salts (to inhibit most G+ve bacteria),

• crystal violet dye (which also inhibits

certain Gram-positive bacteria),

• neutral red dye (which turns pink if the

microbes are fermenting lactose).

 MacConkey,s Media
(Selective and differential media)
• It is used to inhibit the
growth of Gram-
positive bacteria by bile
salts and crystal violet
• selective for G-ve.
 MacConkey,s Media
(Selective and differential media)
• Lactose fermenters form pink
colonies as:
E.coli,
Klebsiella
Enterobacter
 MacConkey,s Media
(Selective and differential media)
• Non Lactose fermenters form
clear colonies as:
Salmonella,
Pseudomonas aeruginosa
Shigella
Proteus,
Yersinia,
Eschericia coli on MacConkey Agar:
growth, with pink colonies
 Mannitol salt agar
(selective and differential medium)
• It contains a high concentration (~7.5%-10%) of salt (NaCl), making it
selective for gram positive bacterium Staphylococci (as high level of
NaCl is inhibitory to most other bacteria).
 Mannitol salt agar
(selective and differential medium)
• It is a differential medium for mannitol-fermenting staphylococci,
containing carbohydrate mannitol and the indicator phenol red (pH
indicator for detecting acid produced by mannitol-fermenting Staph.
 Mannitol salt agar
(selective and differential medium)
• If an organism can ferment
mannitol, an acidic byproduct
is formed that will cause the
phenol red in the agar to turn
yellow (Staphylococcus
aureus)
• Other Staphylococci produce
small pink or red colonies with
no color change to the
medium.
 Mannitol salt agar
(selective and differential medium)
2) Staphylococcus
epidermidis
3) S. aureus colonies
 Mannitol Salts Agar
(MSA)

Coagulase –ve Staphylococci Staphylococcus aureus

 Löwenstein–Jensen medium
• a growth
medium specially
used for culture of
Mycobacterium
 Löwenstein–Jensen medium
• M. tuberculosis
appears as brown,
granular colonies
(sometimes called
"buff, rough and
tough").
 Triple sugar iron media (slant)
• TSI slant is a test tube that contains:
 agar,
a pH-sensitive dye (phenol red),
1% lactose,
1% sucrose,
0.1% glucose
ferrous sulfate .
 Triple sugar iron media(Indicator media)
• Uses: differentiation of bacteria; if organism ferments:
 Glucose only enough acid is produced to turn the butt yellow. The slant will
remain red
 Lactose &/or sucrose  a large amount of acid turns both butt and slant
yellow, thus indicating the ability of the culture to ferment lactose or sucrose.
• Iron: Ferrous sulfate: Indicator of H2S formation
Triple sugar iron media
 Sterilization scheme of media
All by AUTOCLAVE except:
• 3 media by inspissation (70-80 c/2 h/3 days):
Loffler`s serum slope
Dorset`s egg medium
Lowenstein jensen media
• 2 media by steaming at 100 c:
T.C.B.S
Selenite F broth
• 1 media by tyndillization (steaming/30 min/3 days)
Sugar media
 streak method of culture

It is the routinely used EITHER to purify only one bacterial

species (one isolate) from a mixed bacterial culture OR to
check the purity of a bacterial culture containing a single
bacterial species.
 Spread plate culturing Method

• It is used to estimate viable count

Biochemical Tests
 Biochemical Tests for enzymes secreted by
bacteria as:
• Catalase test
• Oxidase test
• Urease test
• Coagulase test
• Nitrate reduction
Biochemical tests commonly used to identify
G+ive Bacteria:
• Catalase Test
• Coagulase Test
• Test for type of hemolysis
• Taxos P (optochin sensitivity testing)
• Taxos A (bacitracin sensitivity testing)
 Biochemical tests commonly used to identify
G-ve Bacteria:
• Oxidase Test
• Indole Test
• Citrate Utilization test
• Methyl Red / Voges-Proskauer (MR/VP)
• Triple Sugar Iron Agar (TSI) test
• Urease test
• Fermentation of glucose, lactose, sucrose and/or manitol
 Catalase test
• This test demonstrates the presence of enzyme
catalase in the organism.
• The enzyme catalase mediates the breakdown of
hydrogen peroxide (H2O2) into oxygen gas and
water  bubbles
2H2O2  O2 + 2H2O
• Positive: Staphylococcus aureus, Bacillus, Entero-
bacteriacae, Gonococcus, Meningococcus, Vibrio
cholerae
• Negative: Streptococcus spp., Clostridium
 Catalase test
2H2O2  O2 + 2H2O

Staphylococcus aureus Streptococcus spp.

Catalase test
Catalase test
CATALASE TEST
 Oxidase test
• It is a test used to determine if bacteria
produces cytochrome c oxidases.
• It uses disks impregnated with a reagent.
• The reagent is a dark-blue to maroon
color when oxidized, and colorless when
reduced.
• Oxidase-positive bacteria (aerobic)
possess cytochrome oxidase or
indophenol oxidase.
 Oxidase test
• Positive with: Pseudomonas spp., vibrio spp., Neisseria spp.,
Haemophilus spp.
• Negative with: E. coli, Enterobacteriaceae, Acenitobacter spp.
 Coagulase test
• This test is used to differentiate Staphylococcus aureus(positive) from
coagulase negative Staphylococci.
– Coagulase Positive (has coagulase enzyme, it will clump the plasma):
 Staphylococcus aureus
– Coagulase negative (does not have coagulase it will not clump
plasma):
 Staphylococcus epidermidis
Coagulase test
Fibrinogen  Fibrin
 Coagulase test

+ve –ve
 Methyl Red/Voges Proskauer (MR/VP)

• Both tests are used to differentiate species of the

family Enterobacteriaceae.
 MR: tests for acid end products from glucose fermentation.
 VP: tests for acetone production from glucose fermentation.
• Media and Reagents Used:
 Glucose Broth
 Methyl Red indicator for acid
 Voges Proskauer reagents (A: 5% Alpha-Naphthol & ethanol, B: KOH &
Deionized Water.
 MR/VP continued

• Reading Results:
 MR— a + result is red (indicating pH below 6) and a – result is yellow (indicating no acid
production)
 VP—A + result is red after VP reagents are added (indicating the presence of acetone) and
a – result is no color change.

Methyl Red: left – and right + VP: left + and right –

 Citrate
• This test is used to differentiate species of the family Enterobacteriaceae.
• It is selective for bacteria that has the ability to consume citrate as its sole
source of carbon and ammonium as sole nitrogen source.

• Media and Reagents Used: Simmon’s Citrate Agar contains

sodium citrate (carbon source), ammonium ion (nitrogen source), & pH indicator
—bromthymol blue.

• Reading Results:
– A + result is blue (meaning the bacteria metabolized citrate and produced an acid end
product) and a – result remains green
 Citrate

positive negative.
 Urease test (Urea Hydrolysis)
• This test is done to determine
a bacteria’s ability to hydrolyze
urea (yellow-orange color) to
make ammonia (bright pink
color) using the enzyme
urease.
• Positive with H. pylori
• Negative with E. coli
 Motility Test
• This test is done to differentiate bacteria
that are motile.
• If bacteria is motile, there will be growth
going out away from the stab line, and
test is positive.
• If bacteria is not motile, there will only be
growth along the stab line.

+ – +
Biochemical reactions of
E.coli
STAPHYLOCOCCI
 Morphology

Gram-positive cocci in clusters

 Staphylococcus

• Why only Staph aureus grew on mannitol salt

agar?
• MSA is selective for G+ve Staph as it contains 7%
salt that inhibits other non- staphylococcus Gram
+ve bacteria and Gram-ve bacteria.
 Staphylococcus

• Why S. aureus and S. pneumonia failed to grow on

MacConkey agar plates?
• MacConkey agar contains the bile salts & crystal violet
which inhibit the growth of Gram-positive bacteria.
Catalase POS

Staphylococcus
Catalase NEG
 S. aureus
• B. hemolysis

• Yellow colonies on
Mannitol salt agar
 Staphylococcus
aureus

Coagulase POS

Coagulase NEG
Streptococci
Describe the morphology of Streptococci
– Gram positive cocci
– Arranged in chains
– Non motile
– Non spore forming
– Facultative anaerobes
– Catalase negative (Staphylococci are
catalase positive)
Freshly isolated Streptococcus
Acute follicular tonsillitis
 Streptococcal follicular
tonsillitis is diagnosed by:

1. Speciemen
2. Smear
3. Culture
Pneumococci
 LStreptococcus pneumoniae
Gram-positive
cocci in pairs;
lancet-shaped
 Streptococcus pneumoniae

• Colony morphology
– Smooth, glistening,
wet-looking, mucoid
– -Hemolytic
– CO2 enhances growth
 Laboratory Diagnosis:
Streptococcus pneumoniae

• Identification
– Catalase negative

– Optochin-susceptibility-
test: susceptible
S.pneumoniae Gram stain
S. pneumoniae
 S.pneumoniae Gram stain
• S. Pneumoniae
Quellung Reaction (Streptococcus
pneumoniae)
Quellung reaction (Streptococcus
pneumoniae)
S.Pneumoniae showing alpha hemolysis on
blood agar
Optochin Susceptibility Test
Optochin resistant
S. viridans

Optochin susceptible
S. pneumoniae
 Bile Solubility test
• Results:
– Positive test appears as
clearing in the presence
of bile while negative test
appears as turbid
– S. pneumoniae soluble in
bile whereas S. viridans
insoluble
Diphtheria
Volutin granules staining of C.diphtheriae
by Albert stain
Volutin granules staining of C.diphtheriae
by methylene blue
Blood tellurite
Loffler’s serum
 Coliform bacilli
1- E.coli 2- klebseilla 3- Citrobacter
Microscopic picture
Pink lactose fermenting colonies on
MacConkey’s
 Mucoid appearance of
Klebsiella on MacConkey’s
 Triple sugar:
yellow slant &
yellow butt
with gas
 IMVC reaction
Indole MR VP Citrate

E.coli + + - -
Klebseilla - - + +
Citrobacter - + - +
 Indole test
-Klebseilla.
-Citrobacter.
E.coli
 Methyl red test
E.Coli.
Klebseilla negative positive
Citrobacter.
 Voges-Proskeure test

E.Coli. Klebseilla.
Citrobacter. negative positive
 Citrate utilization test

Positive = Negative
Blue in: =Green in:
-Klebseilla.
E.coli
-Citrobacter.
 Salmonella
• G-ve bacilli,
• motile,
• non sporing,
• non capsulated
Salmonella colonies with black center
 SHIGELLA ON TSI AGAR
Red slant
Yellow butt
Without gas
 Anthrax
•Gram-positive, spore-
•On Nutrient agar: Medusa head
forming, non-motile bacillus
•On Blood: Non haemolytic
B. anthracis: Gram-positive, spore-forming, non-motile bacillus
Clinical forms are cutaneous, inhalational & intestinal
Growth of the organism on
gelatin agar
 Thiosulfate-citrate-bile salts-sucrose
agar (TCBS)

• Yellow coloured (sucrose-

fermenting) colonies of
Vibrio cholerae on TCBS
agar.
PROTEUS
 Proteus on blood agar

PROTEUS ON BLOOD AGAR

• It grows well on ordinary media giving a
spreading growth in several waves
(swarming).
PROTEUS on ordinary media (agar)
 BIOCHEMICAL REACTIONS
PROTEUS

TSI red slant black butt

with gas production
(H2S producing)

TSI
PSEUDOMONAS
 MORPHOLOGY
Gram negative bacilli,
Actively motile by a few terminal flagella,
Non sporing
Some strains are capsulated.
 CULTURE

• Strict aerobe, grows at 37°C

• On ordinary media, the media turn
greenish blue due to diffusible
exopigments; pyocyanin (blue) and
fluorescin (yellow green).
 PSEUDOMONAS CULTURE

• Most strains produce • On MacConkey's

haemolysis on blood medium they give
agar. yellow pale colonies.
GREENISH-BLUE COLOUR OF PYOCYANIN AND
FLUORESCIN OF PS. AERUGINOSA
PS. AERUGINOSA
 PSEUDOMONAS AERUGINOSA
Gram stain of P. aeruginosa.

P. aeruginosa on Mueller-Hinton
agar plate. The organism
produces pyocyanin, which is
blue, and pyoverdin, which is
green. Together these pigments
produce the blue green color that
is seen in the agar around the
pseudomonas growth.
Key identification characteristics for
Enterobacteriaceae
Mycobacterium TB
 Staining
• They are acid and alcohol fast as demonstrated
by Zeihl- Neelsen technique of staining.
• They appear as thin pink rods, single or in small
group against blue background.
• Using flourochrome stains (e.g.) auramine and
examined under ultraviolet microscope, it
appears as golden yellow rods against a dark
background.
Mycobacterium T.B (Z.N stain)
 Mycobacterium T.B
Acid-fast bacilli in a Z.N stain of sputum
Mycobacterium T.B on LJ media
Mycobacterium T.B on LJ media
Tuberculin test
Gram-negative
Diplococci
Neisseria
N. meningitidis
N. gonorohoea
commensal : N. pharyngis
Pathogenic Neisseria in pus
(G-ve diplococci)
Pathogenic Neisseria
(G-ve diplococci)
Gram positive flowchart
Gram negative flowchart
Antigen-antibody
reaction
 Types of Ag-Ab Reaction:

• Agglutination
• Precipitation
• Antibody labeled assay
• Complement fixation
• neutralization
 Types of agglutination

1- Direct agglutination
2- Passive agglutination
3- Antiglobulin agglutination
4- Coagglutination (CoA)
5- Virus Haemagglutination inhibition
6- Heterophile antibodies agglutination
 Direct agglutination:

a) Qualitative (Slide method):

 Blood grouping.
 Typing of bacteria
b) Semi-quantitative (Tube method)
Slide agglutination test
-VE +VE

+VE
Identify: Slide hemagglutination test
Result: +ve test
Enzyme-linked immunosorbent assay
 Quantitative hemagglutination test

• Define titre?
 Quantitative hemagglutination test

• Define prozone effect?

• The lack of agglutination at high concentrations of
antibodies is called the prozone effect. It is due to Ab excess
 Indirect Coomb's Test

• Application: To see if the mother has anti-Rh

antibodies in her serum.
 Direct Coomb's test

• Application: Antibodies to the Rh factor generally do not agglutinate

red blood cells. Thus, red cells from Rh+ children born to Rh-
mothers may be coated with anti-Rh Abs from the mother. To check,
a direct Coombs test is performed.
 Single radial Immunodiffusion

• It is a quantitative test, commonly used in the

clinical laboratory for the determination of
immunoglobulin levels in patient samples.
 Immunoelectrophoresis

• This test can also be used to evaluate purity of

isolated serum proteins.
Antibiotic Sensitivity Testing
Antibiotic Sensitivity Testing
(Serial Dilution Method)
 Antibiotic Sensitivity Testing
(Disk diffusion Method (Kirby-
Bauer procedure)
 Antibiotic sensitivity test

• Minimum inhibitory concentration (MIC) is the lowest

concentration of an antimicrobial that will inhibit the visible
growth of a micro organism after overnight incubation
Series dilution method The disk diffusion method
The zone of inhibition guides the right
choice of Antibiotic
virology
 Cytopathic Effects of viruses

Hemadsorption Nuclear
pyknosis
Cytopathic Effects of viruses

Syncytia formation
HEMADSORBTION

add red blood cells

Cytopathic Effects of viruses

RSV (cytoplasmic inclusions)

 Cytopathic Effects of viruses

Rounding & detachment of tissue culture cells

due to their killing (Herpes, vaccinia viruses
 Cytopathic Effects of viruses
• Inclusion bodies: are
visible sites of viral
assembly or cellular
damage as:
• Negri bodies of rabies
virus
 Cytopathic Effects of viruses

Enlarged, aggregated and ballooned cells causing

multi- nucleated giant cells
(herpes giant cell virus
Mycology
 Morphology
• Two morphological forms of fungi are observed:
– Yeast
– Hypha.
a. Hypha(septate, non-septate) b. Mycelium c. Yeast form, budding
C. albicans on Sabouraud's dextrose agar

typical cream coloured, smooth surfaced, waxy

colonies.
Candida albicans
Chlamydospore production by C. albicans
 Germ tube test
• Method: grow in serum for 3 hours at 37°C; make
a wet film and examine for formation of
filamentous outgrowth (germ tubes).
CASE

• A 43-year-old male with AIDS is referred to your clinic to

be evaluated for painful cracks at the corners of his
mouth, which he had for two days.
What is your Clinical diagnosis?
• Angular stomatitis
Other clinical presentation

Cutaneous candidiasis

Onchomycosis
Cells of innate immune response