Importance of microscope

Magnification: It is degree of enlargement. It means produce

an enlarged image of the object that are too small to be seen
by naked eye

Resolution: The ability to distinguish detail

or ability of a lens to separate or distinguish small objects that
are close together
 Types of microscopes

A) - Light microscope
➢ Bright field microscope (compound Microscope) (ordinary student
microscope).
➢ Dark field microscope (dark ground illumination).
➢ Fluorescence microscope.
➢ Phase contrast microscope.
➢ Stereoscopic microscope

B) - Electron microscope
A) - Light microscope
1- Bright field microscope (compound microscope)(ordinary microscope)(student
microscope)(most common)

Parts of the compound microscope :

Base: Supports the microscope and used for carrying the microscope.
Arm: Supports the body tube and is used to carry the microscope.
Light source (illuminator): Provides light (source of light)
Condenser: It is system of lenses which collect or condense the light and
direct it to the slide and not magnify image. It can be moved up and down by sub
stage adjustment. For bacteriological work, condenser must be highly raised to
collect the largest amount of light
Iris diaphragm: It present in the condenser. It controls the amount of light. For
bacteriological work, iris must be widely opened.
Stage: Used to support the slide.

Stage clips: Used to hold the slide in place.

Coarse adjustment knob: Moves stage up and down a large amount to focuses the
image under low power.

Fine adjustment knob: Moves stage up and down a tiny amount to sharpen the
image under all powers.

Q&A : to focus the image we use coarse adjustment Knob, while to

sharpen the image we use five adjustment knob

Draw tube: Connects the eyepiece to the nosepiece. For the majority of the
microscopes, draw tube length is 160 mm.

Nosepiece: Holds all the objectives and it revolves.

Ocular lens or eyepiece: Magnify image formed by objectives (10X) fits into a
graduated draw tube which fixed on the body tube.

Objective lenses: Magnify the image. There are four objectives with different
magnifying powers
 Objective lenses

Type of objective Magnification Uses

scanning 4 times for getting a general overview of a slide.
Low power 10 times in parasitology
High power 40 times in histology, pathology
Oil immersion lens 100 times in bacteriology
The oil immersion lens has the highest magnification (100X) and the highest
numerical aperature (1.25) or the highest resolution

The numerical aperture (NA): the widest cone of light that can enter the lens Or
the ability of the lens to gather light and show fine specimen detail (the more the
numerical aperature >> the more the resolution of the microscope)

What is meant by10x / 0.25

•10x: it magnifies the object 10 times.
0.25 is N.A. of this lens. •
 Oil Immersion Lens (OIL)
Cedar wood oil must fill the space between the objective and the
specimen on the glass slide as the space between the objective
and the slide has air which has RI less than glass which leads to
refraction of light rays away from the lens.

Q&A
Why must use oil immersion lens (oil)?
as oil has the same refractive index as glass (1.5) >>so no refraction of
light rays occurs>>>> so all light rays will enter the lens opening >>>so
give high clearance and resolution of the image.

Density=1.5
dense medium Density=1.5

Light rays are refracted out rays not refracted

 Magnification

Enlargement of image too small to be seen by naked eye

Total magnification= objective lens Mag x ocular lens Mag

Q&A the total magnification power of the microscope using oil

immersion lens is 1000X while the magnification power of oil
immersion lens alone is 100X
‫ألن‬
Total Mag = objective lens Mag (oil immersion lens Mag) x ocular lens
Mag(10 ‫= (قيمة ثابتة تساوي‬100x10=1000X
Factors affecting magnification
Draw tube length
Focal length of objective lens
Eye piece magnification
Wave length of light (The shorter the WL the higher the
magnification)
 Resolution Resolving power

The ability to distinguish detail

It depends on the wavelength of light used and on the numerical aperture (NA).

The shorter wavelength  the greater resolution the greater magnification

The higher (NA)  the greater resolution

Contrast
Def :Differences in intensity between object & background
NOTE: Even with sufficient magnification and resolution, you can visualize an object under
a microscope only if there is sufficient contrast between the object and its surroundings

Q&A you can improve contrast using stains and by regulating the
opening of the diaphragm
1. Image is too dark! - diaphragm/ light source
2. There's a spot in my viewing field, even when I move the slide the spot stays in the same
place! Your lens is dirty. May be dust.
3. I can't see anything! Blurred image- Coarse adjustment, oil must touch lens , then fine
adjustment
4. Only half of my viewing field is lit, it looks like there's a half-moon in there!. objective not
fully clicked into place.
5- Type of Lenses in student microscope

6- Microscope Care
1- Always carry with 2 hands
2- Don’t touch the lenses with your fingers, Only use lens paper for cleaning
3- Do not force knobs
4- Always store covered& leave the microscope with the low power objective in place.
Q&A what are the Proper Use of the Microscope with oil-immersion lens
(Precautions during using OIL) ?
The condenser must be highly raised.
The iris must be widely opened.
Cedar wood oil must fill the space between the objective and the
specimen
Sterilization: It is absolute process of removal or killing all
living microorganisms including bacteria and their spores

Disinfection: removal or killing most pathogenic

microorganisms (not necessarily all microorganisms

(Reducing the number of pathogenic microorganisms to the

point where they no longer cause diseases
▪ can not kill bacterial spores

Q&A
Killing all M.O including spores is sterilization, while reducing the
number of M.O is disinfection
.
 sterilization

chemical physical
• Heat (moist heat
& dry heat)
• Filtration
• radiation
 Physical Methods of Sterilisation
Sterilization By Heat: Of the
Charring ‫حرق‬ essential
1-Dry heat M.O.A Oxidation ‫ اكسدة‬components
of M.O
(reversable)

Red heat Flaming infra red

Bacteriological loop Mouth of test tube and flask Surgical instruments
Points of forceps Slides and cover slips and Surgical rooms
Inoculating wires Scalpels
Hot air oven:
Glass wares(test tube&petri dish ‫أي حاجة زجاج سواء‬
Surgical instruments
Dry materials in sealed containers as talc powder
&Oil Sterilization time: 1 1/2 – 2 hours at 170 – 180 °C
1.5 hr>>>200 ̊c & 2 hr>>>170 ̊c Q&A the mouth of test tube is sterilized by flaming,
while the test tube is sterilized by hot air oven
 Moist heat
(irreversible)
M.O.A

Coagulation and denaturation of bacterial proteins

which is irreversible
1-Moist heat below 100°C

Pasteurization Tyndallization
Plasma
For milk
Holder method: 63°C for ½ hour Serum
Flash method: 72°C for 20 minutes
Ascitic fluid
UltraPasteurization: 82°C for 3 minutes

At 56°C in water bath for an hour

on seven successive days

Followed by rapid cooling

 2-Moist heat at 100°C:

Boiling Free steaming

Q&A (Koch`s sterilizer)

For 5-10 minutes (Koch`s steamer)

Using distilled water and 2%
Na carbonate to avoid rusting
At 100°C for an hour
Used for:
Used for:
Glass wares
Litmus milk media
Surgical instruments
Sugar media
Rubber stoppers
Gelatin media
 3-Moist heat above 100°C
(steaming under pressure)
Autoclaving)

Q&A
Sterilization at 121°C for 20 minutes 15 lb/sq. inch
Used for:
All bacteriological media EXCEPT??
Litmus milk &sugar& gelatin
Condemnation of media
Surgical instruments wrapped with
apiece of cloth
Surgical supplies as dressing, towls,
linen, gauze
 Sterilization by filtration
Used for: heat labile solutions as :Plasma, Serum, Ascitic fluid, vitamin C , urea solution and
antibiotic solutions
Filtration not kill M.O but just make surgical removal
Types of bacterial filters:
1-Earthenware candle
2-Sintered glass filter
3-celluse membrane filter
4-Seitz filter

Test of efficiency
Using Serratia marcescens m.o.
System of bacterial identification
1-microscopical examination for detection of
morphology

2-culture character
3-biochemical tests
4-serological test
5-lab animal inoculation
6-antibiotic sensitivity test
7-molecular diagnosis (PCR&DNA sequencing)
Light microscopes are the most
commonly used microscopes.

There are two basic types of

preparation used to view specimens
with a light microscope:
1) wet mounts
2) flame fixed specimens
 To heat-fix a sample, a thin layer of the specimen is
spread on the slide (called a smear)
Smear preparation and fixation

NB: If the smear is too thick, proper decolorizing will not be possible.
If the smear is overheated during fixing, the bacterial cell walls will rupture
 Staining
differential staining
Simple staining (use of 2 contrasting stains Special
(use of a single stain) separated by a decolorizer)

Gram stain (ZN) stain

Basic Acidic
(Positive) (Negative) Acid fast Non Acid fast
GPB GNB
stain stain
violet red
Alc fast Non Alc fast
Acc to ability to resist
decolorization by alc
 Differential staining
1- Gram Stain
Def: Differential stains use two stains.
Purpose:
1-To view cellular morphology
2-Differentiating bacterial species into two large groups
(Gram-positive and Gram-negative) based on of their
cell wall.
 Reagents Q&A
Come In And Stay‫مهم بالترتيب‬
1-Crystal Violet (violet) (2min) Primary stain;
positive stain (Stains cell wall purple) Then Wash
2-Iodine (brown)(1min) Mordant
(Helps to fix the primary dye to the cell wall)
it combines with CV to form an insoluble complex that gets trapped
in thicker peptidoglycan layers Then Wash
3-Alchoal (95%) (5sec) Decolorization (it is the most important step)
(the differential step) why? Because overdecolourization >>>gram
positive bacteria lose its stain and appear red
CV-I complex washed out of Gram negative organisms because it
cannot be trapped by peptidoglycan layer; flows right through outer
membrane Then Wash
4-Safranin (pink) (2min) Counterstain or secondary stain
positive stain that provides contrasting dye for decolorized cells
(Gram negative) Then Blot Dry With Bibulous Paper Then Add Cedar
Oil Then Examine under Oil Immersion Lens
 Gram Staining Procedure
1
2 3
Wash with Wash with Wash with
tap water tap water tap water

2 min
1 min 5 sec

4 5 66
Add cedar oil
Wash with
tap water

2 min Blot dry with Examine under

bibulous paper oil immersion
lens
 cell wall
Gram-positive Gram-negative
lipid Low ( no O.M.) lipid high (O.M.) (LPS)
Alc dissolve lipid---- small pores Alc dissolve lipid---- large pores---not closed
Alc dehydrate--- shrinkage---tightening--- on dehydration--- incease permeability
closure pores----reduce permeability
PPG Thin (5-10% )
PPG Thick (90% ) unable to retain CV- Iod complex
crosslinking ----multilayers ---trapping/
retention CV- Iod complex

MgRNA + C.V- iod------larger insol

complex Because of

CV-I complex tightly bound CV-I complex unbound

not washed out washed out
Colorless cell

counter stain not absorbed counter stain absorbed

counter stain not absorbed
Q&AWhy?
Cell remain violet Cell appear red
 Results
 The standard abbreviations for the four types of Gram
stain and morphology are:
1. Gram Positive Cocci (GPC) as staph
2. Gram Positive Bacilli (GPB) as E.coli
3. Gram Negative Cocci (GNC) as Neisseria
4. Gram Negative Bacilli (GNB) as bacillus anthracoid
 Q&A

Gram’s +ve Cocci arranged in Gram’s – ve Cocci

grape like clusters arranged in Diploid
27
Staphylococci Neisseria
. Q&A

Gram’s –ve bacilli Gram’s +ve bacilli arranged in

E-coli chains
Bacillus anthracoid
➢ factors affecting gram staining
Conditions should be followed for a valid Gram staining
procedure or
Young cultures - must be young within 18-24 hrs old
(older cultures lose their Gram staining properties due to
changes in the CWs as the cells get older)
Thicker or uneven smears will result in uneven staining
and decolorization.
Overheating cells during fixation
Fresh reagents - of proper strength
Proper order of adding the reagents
Proper time for each stain and for decolorizer as overdecolourization
remove the stain of gram +ve bacteria and appear red
Q&A staph under microscope >>>red cocci ….explain?
Due to over decolourization or aging of the bacteria
 Potassium hydroxide test (KOH test)
Used to quickly distinguish between gram -ve and gram +ve bacteria as a complement to Gram
staining.
principle
KOH dissolves thin PG layer of gram –ve bacteria, cell walls lyses and release its contents (DNA).
DNA make the solution viscous and stick to the loop .
Gram +ve bacteria not affected by KOH, because they have thicker PG layer in cell wall. No lyses
to cell wall, no release DNA and no viscosity observed.
Method
Apply drop of 3% KOH on a slide. Transfer bacteria (cultivated for 24-48 h) to the drop of KOH.
Stir carefully for 30 sec
Result
Positive results: viscous solution (gram negative)
Negative results: no viscous solution (gram positive).
 Types of culture media
I- Based on their consistency:
a) Solid medium: (2-3% agar agar were added)
*Agar agar ( func.: solidifying agent )is:
polysaccharides derived from seaweed, solidifying agent,
non nutritive ( bacteria can not utilize it ) dissolved by
boiling at 100 and solidified at 45)
b) Liquid medium: no agar agar
c) Semi solid medium : 0.5% agar

II- Based on the constituents/ ingredients

a) Simple medium
b) Enriched medium
c) Enrichment media
d) Selective medium
f) Differential and indicator medium
 Simple (Basal) media
Def: Media consists of only basic necessities for growth.
Use: cultivation of non-fastidious bacteria (all except Strept (on
enriched media as blood agar ), coryne , nisseria, hemophylus)
micobacterium
a- Peptone water: peptone 1% +NaCl 0.5 % +
D.W used as a base for sugar media + indole
test. Example:
b-Nutrient broth (NB): peptone water + beef
extract
c-Nutrient agar (NA): NB + 2% agar agar
d-Gelatin media: NB + gelatin+ 0.5%
 Mannitol salt agar (MSA) ‫الجدول كله مهم‬
MSA Selective For staph Inhibitor: Pathogenic staph: yellow
high NaCL salt halo around colony (MF). S.
(7.5 %) aureus

Differential Between staph into Indicator: Nonpathogenic staph: pink

pathogenic staph and phenol red colony(NMF). S.
non-pathogenic staph Epidermidis

Mannitol
Non fermenter

Mannitol
fermenter
 MacConkey’s medium ‫الجدول كله مهم‬
Media type to Due to Result
for (inhibitor) LF (Pink Colonies):e-coli,
MacConkey Selective enterobacteriacea Bile salt klebsiella, citrobacter,
enterobacter.
Between (indicator)
Differential Neutral red+
enterobac. Into LF NLF (pale Colony):
(sugar) lactose
and NLF salmonella, shigella, proteus,
providence,morganella

pale Colony

Pink Colony
 Eosine Methylene blue (EMB)
‫الجدول كله‬
‫مهم‬
EMB Selective for (inhibitor) SLF ( metallic
enterobacteriacea eosin,methylene blue green sheene):-
Differential coli, citrobacter
between LF into (indicator) eosin,
strong lactose F methylene blue which
MLF ( fish eye
and moderate F inhibit Gram +
bacteria, thus colony):in12hr
favoring growth of … klebsiella appear
pink to violet
 ‫الجدول كله مهم‬ Blood agar
Enriched and Differential: Extra nutrients: Blood Indicator: RBCS
Alpha hemolysis: Partial lysis Beta Hemolysis: Gamma or no hemolysis: No
of RBC (a greenish zone Complete lysis of RBC hemolysis of RBC. No change
around the colony) due to the (clear zone around colony) of the medium under and
reduction of RBC hemoglobin surrounding the colonies.
to methemoglobin by
hydrogen peroxide produced
by the bacterium,
 Why cultivate bacteria?
Indications/ Need for culture:
➢ Isolation of Microorganisms from samples
➢ Obtaining pure cultures
➢ Colony characteristics

• To estimate viable count.

• To test antibiotic sensitivity
➢ Typing bacterial isolates

Biotyping: Morphology and staining reaction

Biochemical reactions
Serotyping: Serology
➢ Lab. animal pathogenicity
➢ Bank strain for future use including vaccine
 Sterile Technique
Aseptic Technique = procedures that minimize the introduction of microorganisms to
media (cultures) or from cultures to surrounding environment
The following conditions must exist for aseptic technique to be successful:
1. The work area must be wiped with an antiseptic to reduce the number of potential
contaminants.
2. The instruments must be sterile.
3. The work must be done quickly and efficiently to minimize the time of exposure during
which contamination of the culture or laboratory worker can occur.
4. Work near the flame by at most 15 cm as the M.O can not resist the hot air in this area
How is media made?
• Accurately weigh dry powdered nutrient media into bottles, add distilled water, cap

it and autoclave (121°C at 15 psi for 20min) Q&A all medias (as MSA media

)are sterilized in autoclave except litmus milk, sugar , gelatin media are
sterilized in KOch’s steamer
• Once the media is autoclaved it is sterile (all m.o killed).

N.B
• when save the petri dish in refrigerator it must be inverted to avoid condensation
• To make sure that the petri dish is sterile before cultivate on it >>>> make sterility test
When the temperature of the bottle reaches 50°C, quickly
proceed as follows

1- Hold bottle in your hand, Remove the rubber stopper & flame the bottle mouth
3- Partially lift the cover the dish and Pour about 20 ml of media to cover at least 2/3
of the plate surface
4- Gently swirl the plate to spread media on all surface (about 3-4 mm depth)
5- Close the plate cover, wait until media solidifies without moving the plates.
Having poured the media at low temperature (50°C), very little condensation will
form on the inner side of the cover
6- Turn dishes upside down (media on top, cover on bottom).

From this moment on, plates should always be kept upside down
 Aseptic incubation
Sterility test
is designed to demonstrate the presence or
absence of contaminating microorganism in
plates before using it..

Incubate plates at 37 for 24hr .it must be sterile

no growth observed.
 Deep agar
Broth (liquid) Slant agar (solid)
(semi-solid)

‫الصور هتيجي ف االمتحان وهيسأل‬

‫على االسم واالستخدام‬

Agar plate (solid)

 broth cultures
Q&A
Use : Liquid medium is best when you want to rapidly
increase the concentration of the organism.

Result

Dis: Cant see colony morphology.

Cant separate organisms from a mixed culture (Don't provide a
pure culture from a mixed culture)
 Semi-solid media ‫الصور مهمة‬
(deep agar)
Stab culture =Deep inoculation
Use: This method is used to study the ability of bacteria to:
1. Motility
Q&A
2- Oxygen requirement (Grow with the presence of O2 or not )
3- gelatin liquefaction (Gelatin media) Deep agar

Method:
Sterilize the needle (until red hot) → cool
→ colony →stab in middle and remove
via same stab
Do not use a loop
 Result:
Motile: diffuse growth (e-coli)
Obligate aerobes
Non motile: growth restricted on
inoculum site (staph)

‫الصور مهمة‬ Facultative anaerobes

Obligate anaerobes

Microaerophile
Non
Motile
motile
 Solid media (slant
agar) Q&A
1-Slant culture or stroke culture Why agar
slants better suited
The medium allowed to solidify at an angle in order to get a flat than agar plates to
inoculating surface maintain stock
cultures?
Use: As slant agar is
Space saving
-Preservation (short preservation in refrigirator 1week) Dehydration and
-Space saving contamination are
-For serological test. faster in agar plate
Method: than slant agar

‫الصور مهمة‬
 2-Agar plate

Streak plate Spread plate Pour plate

Use -The streak plate provide a uniform surface Use
method allows Isolation growth (Lawn or carpet
Gives an ➢
of bacteria in pure culture culture)
estimate of
(a single colony ) the viable
Use
Identification of colony ➢ bacterial
morphology Antimicrobial sensitivity ➢ count in a
test suspension.
separate organisms in a ➢
bacteriophage typing ➢
mixed culture
To quantitate ➢
identify the organism: ➢ bacteria in
biochemical tests to urine
identify bacteria are cultures.
only valid when
performed on pure
cultures
 with or without firing
A) Streak plate method

Streak plate
B)- Spread plate (Lawn or carpet culture)
C)- Pour plate method
Pure culture Mixed culture

Originate from one strain Originate from two or more strain

All colonies look the same colonies have different size and shape
Subculturing
Bacteria Preservation Q&A
 Q&A
why Passing the mouth of a tube through the flame of a Bunsen burner?
This prevents airborne contaminants from entering the tube.

Why we should Partially lift the cover of the first dish?

To avoid contamination

How much Agar do you need in a petri dish? Pour about 20 ml in Petri dish.
If too little , not be enough to cover the dish &the plate will dry up easily).
If too much, the cover dish will come in contact with the nutrient agar, leaving no
room for microbial growth, wasting media).

Why are agar plates stored upside down( inverted)?

because moisture condenses on the lid and will drop down onto the agar/growth
medium causing moisture problems and interfere with the developing microbes
Slope cultures in screw cap bottles are preferred to maintain a stock of a pure
culture ? Slants are better suited because they can be capped, preventing
the agar and culture from drying out. The cap also prevents airborne contaminates
from entering the slant. Slants take up less storage space.
Slope cultures are preferred to broth (i.e. liquid medium) cultures because the first
sign of contamination is much more readily noticed on an agar surface.

To transfer cultures from one medium by inoculating another medium. This is called
subculturing
viable plate count ( Colony Counting) : ‫هييجي مسألة‬
Colony counting after plating dilutions of the sample onto growth
medium.
Countable number of colonies= 30 - 300 per plate
Why ? because
less than 30 colonies, small errors in dilution technique or the presence of
contaminants will effect on the final count. Likewise, if there are more than 300
colonies colonies will have grown together.
• You count 46 colonies on your plate.
• You put 1 ml of bacterial culture into 99 ml of saline and
plated 20 µl.
• Calculate TCC.

Test your self

TCC = No. of colonies x reciprocal of dilution Cfu/mL
Plated volume (mL)

1ml=1000µl
 Qualitative method

Kirby-Bauer Test
(disc diffusion test) (agar diffusion method)
(antibiotic sensitivity test)
Principle:
an antibiotic disk is applied to the surface of an agar
plate containing the organism to be tested and the
plate is incubated at 37°C for 24-48 hours.

The effectiveness of antibiotic is shown by the presence

of inhibition zones. These zones of inhibition appear as
clear areas surrounding the disk.

62 11/29/2022
Material and Preparation
1- Mueller Hinton agar (MHA) why? As it
It supports satisfactory growth of most non fastidious pathogens. NB If fastidious
bacteria as sterpt. >>>>use blood enriched agar media
Low in thymine/thymidine (sulphonamide and trimethoprim inhibitors).
Low in Mg & Ca (Aminoglycosides and tetracyclins inhibitors). Good diffusion.
Standard PH and Standard electrolytes

2- standard inoculum size: Inoculum should have turbidity equivalent to 0.5

McFarland standard. Should be from a freshly overnight growth.

3- 0.5 McFarland standard: McFarland standards are used as a reference to adjust

the turbidity of bacterial suspensions so that the number of bacteria will be within
a given range (1.5 x 108 CFU/mL). 0.05 ml of 1% BaCl2 and 9.95 ml of 1% H2SO4.
spread ‫بنفرد ع الطبق بطريقة ال‬
flooding ‫او طريقة ال‬
4-antibiotic disc: stocks can be stored for one month at -20
They are packaged in spring-loaded cartridges containing 25 or 50 disks

63 11/29/2022
Measure the diameter of clear zone around the disc. (Inhibition zone).
at the widest diameter and measure from one edge of the zone to the
other edge using a ruler or calipers

64 11/29/2022
8- Use standard tables (CLSI) to assess if zone size
indicates resistance or sensitivity to that antibiotic.
‫هتيجي مسألة ف االمتحان هيديني ارقام واقارنها بالجدول‬
‫ وال‬resistant ‫ وال‬sensitive ‫وأقول البكتريا كدة‬
antibiotic ‫ ألي نوع‬intermediate

65 11/29/2022
9- Interpretation of results
• Susceptible ”S” :organism is inhibited by antibiotic
Intermediate “I”: organism may require a higher antibiotic dose to •
be inhibited
Resistant “R” :organism is not inhibited by antibiotic •

66 11/29/2022
 Sugar fermentation test
Media: peptone water
Indicator: phenol red that give yellow colour in acidic media
and red color in alkaline media
‫احنا بنحط السكر ف الميديا‬
Lactose sugar
Purpose: dif. Bet Enterobacteriaceae into lactose fermenter(E.coli, Klebsiella,
Citrobacter, enterobacter) that appear yellow and Non lactose fermenter
(salmonella, shigella, proteus) that appear red

The positive result is indicated by:

➢Change of the colour into bright yellow color
(positive result due to acidic product resulted from
fermentation of sugar ) that indicates the production
of enough acid products from fermentation of the
sugar to drop the pH of the media and if the bacteria
not ferment sugar >>>ferment protein (peptone)
>>>alkaline product >>>>red colour>>>negative
result
➢ gases is detected inside the inverted Durham`s
tube
 Triple sugar iron agar (TSI)
USE: used in the differentiation of Enterobacteriaceae by their ability to
ferment glucose, lactose, and sucrose, gas production of H2 , co2 (this is
detected by Fracturing or lifting of agar from base of culture tube)
and produce hydrogen sulfide (H2S) (black colour)
Media: TSI Agar
poured in tube in form of slant with deep butt
and the sugar added is lactose, glucose, sucrose
It contained triple sugars:
(1% lactose, 1% sucrose, 0.1% glucose)
+ sodium thiosulphate
ferrous sulphate (indicator for H2s)
+ phenol red indicator
 Q&A
R/Y or K/A red slant Yellow butt due to glucose fermentation , no
lactose or sucrose fermentation with alkaline slant due to production of
amine’s from protein as shigella
R/Y or K/A with Black ppt: due to glucose fermentation only , no lactose
or sucrose fermentation with H2S production as salmonella,proteus
Y/Y or A/A and gas Yellow butt and slant due to glucose as well as
lactose and/or sucrose fermentation &Fracturing or lifting of agar from
base of culture tube due to hydrogen gas production as Ecoli , Klebsiella
Y/Y or A/A Black ppt: acid butt and slant with glucose and lactose
and/or sucrose fermentation with H2S production as citrobacter

R/R or K/K = no sugars fermentation and utilize peptone as

pseudomonas aeruginosa

Q&A
We can detect gas production in sugar fermentation test by
durhums tube while in TSI by fracturing or lifting of agar
 E.Coli & pseudomonas Salmonella & shigella
citrobacter aeruginosa
klebsiella proteus

Y/Y + gas Y/Y+H2S R/R R/Y+H2S R/Y

NB/ CO2 and H2 gas is detected in sugar fermentation byDurham's tube
…………………….while in TSI by
Fracturing or lifting of agar
………………..
citrobacte
What is meant by Y/Y and black ppt………………….
NB
• We can dif bet salmonella & shigella \ proteous & shigella with TSI but we can not dif.
Bet. Salmonella& proteus with TSI but with urease test as proteus give pink color and
salmonella give yellow color

• We can dif bet E.coli & Citrobacter \ klebsiella& Citrobacter but we can not dif. Bet.
E.coli & klebsiella
 Indole test Citrate utilization test
Media: Treptophan broth Media: Simmon`s citrate agar media

Indicator: Kovac`s reagent Indicator bromothymole blue

Indole+ve: Pink ring Citrate +ve: blue colour

Indole–ve:Yellow or brown Citrate –ve: green colour

ring or no change
 MR-VP test

Media: buffered glucose peptone broth)

mixed acid fermentation ‫>>>>>ال‬pathway ‫الهدف هو معرفة البكتريا هتمشي ف انهو‬
pathway
protein fermentation pathway ‫وال ال‬

If mixed acid fermentation pathway>>> methyl red +ve (red color) & Voges
Proskauer -ve (yellow)

If protein fermentation pathway >>>then voges proskaur +ve (red) & methyl
red –ve (yellow)
 MR-VP test
Methyl Red (MR) Test name Voges-proskauer (VP)

indicator Barritts
Methyl Red
indicator reagents
indicator

result
M.R. +ve: Red colour Voges-proskauer +ve: red
M.R. –ve: Yellow colour Voges-proskauer –ve: yellow
E- coli❖ ‫هام جدا‬ klebsiella

salmonella❖ shigella
 Urease test
Testing the ability of microorganism to split urea into ammonia (NH3)
& CO2.(
Christensen’s urea medium:
pH indicator phenol red
Result

positive: bright pink (fuchsia( ❖

Proteus, klesiella ❖
Negative: yellow color ❖
E- coli , salmonella , shigella ❖
 Q&A

Pink colony on mackonky (lactose fermenter •

(E.coli, klebsiella, Citrobacter)) and y\y on TSI
(E.coli , klebsiella) and fuchsia on urease test(
klebsiella, proteus) so the suspected M.O is
>>>>klebsiella

Yellow on urease>>>E.coli•
Q&A
• Pale colony on mackonky (non lactose
fermenter(salmonella, shigella, proteus)) and
R\Y + H2S on TSI( salmonella, proteus) and
fuchsia or pink colour on urease
test(+ve)(proteus, klebsiella) so the
suspected M.O is>>>> proteus
but
• Yellow on urease(-ve)>>>salmonella
 Oxidase test
Use: differentiate between the families of Pseudomonadaceae
(ox +) and Enterobacteriaceae (ox -)(both are GM-ve bacilli under
microscope) but on mackonky pseudomonas and some
enterobacterecae>>>>>pale colour
Result:

+ve: blue color within

20 sec

-ve: no color
 Q&A
• Gram –ve bacilli under microscope , pale
colony on mackonky , oxidase test positive(
blue color)>>>>pseudomonas
• Gram –ve bacilli under microscope , pale
colony on mackonky , oxidase test
positive(blue color) , on TSI R\R
>>>>pseudomonas
• Gram-ve bacilli under microscope, pale
colour on mackonky , oxidase test negative (
no color) >>>enterobactericae