Dr.Taghreed Khudhur Mohammad
Ahmed Dawood
Lab. 1
2015
Sterilization ‫التعقيم‬
Sterilization: is a freeing of an article from all living microorganisms
including bacteria and their spores, viruses, yeasts, molds (pathogenic
and nonpathogenic)
.‫هو خلو االدوات من االحياء المجهرية المتضمنة البكتريا‬
)‫وسبوراتها والفايروسات والخمائر واالعفان ( الممرضة والغير ممرضة‬
Methods of Sterilization :- ‫طرق التعقيم‬
 Physical methods . ‫الفيزياوية‬
 Chemical methods . ‫الكيمياوية‬
 Mechanical methods ‫الميكانيكية‬
1
sterilization
mechanical
physical
chemical
other
filtration
pH,osmotic pressure ,
highly movement speed ,
sonication
ionizing
gases
formaldehyde
antiseptic
radiation
disinfectant
Non ionizing
heat
Moist heat
Dry heat
Red heat
Temp. above 100 C
Flaming`
Temp. at 100 C
autoclaving
incineration
Temp below 100 C
Hot air oven
tyndalization
boiling
pasteurization
2
Physical methods of sterilization :A -Heat sterilization ‫التعقيم بالحرارة‬
1) Dry heat sterilization ‫الحرارة اجافة‬
a. Red heat used to sterilize wire loops ,point end of forceps
‫الحرارة الحمراء تستخدم لتعقيم الناقل ونهايات المالقط‬
b. Flaming: used to sterilize mouth of tubes , glass spreaders
(which are flamed in ethanol ). ‫األلهاب تستخدم لتعقيم فوهات االنابيب‬
c. Incineration ‫الحرق‬:used in pathological fuming materials .
3
d. Hot air oven ( 160-180 ) ˚C for 2-4 hr., used to
sterilize glass wares ( pipette ‫ ماصات زجاجية‬, syringes
‫سرنجات زجاجية‬, flask ‫ دوارق زجاجية‬, glass Petri
dishes ‫ اطباق بتري زجاجية‬, ….etc ) . ‫فرن الحرارة الساخن‬
,Conical flask , volumetric flask , Beaker, Graduated cylinder , Petri dish
4
2) Moist heat sterilization ‫الرطبة‬
a. Temperature below 100˚C, pasteurization (63˚C for 30 min
) , to sterilize milk .
‫ م لتعقيم الحليب‬100 ‫حرارة أقل من‬
b. Temperature at 100 ˚C
 Boiling (5-10 min ) to sterilize rubber tubes , glass
syringes ( kills all non-spore forming bacteria ) .
 Steaming ( tyndillization ) steam 30 min for 3 days
,used to sterilize gelatin media , sugar media . ‫بخار بدرجة‬
‫ م لتعقيم االوساط الجيالتينة واالوساط السكرية‬100
 C .Temperature above 100 ˚C ( autoclave ) the condition used in this
instrument (121˚C ,15 min, 15 p/inch2 ),used for sterilization of :
 surgical tools and clothes ‫االدوات الجراحية ومالبس العلميات‬
 culture media and to sterilize inoculated media ‫االوساط الزرعية‬
 Swab ‫المسحة‬
 D.W. ‫ماء مقطر‬, Solutions sealed in containers ampuls, vials ,
 Bulk Solutions ‫محاليل بحجوم كبيرة‬
 Glassware
 Instruments Intraoperative sterilization of metallic devices ‫اجهزة معدنية‬
 metallic surgical instruments
5
B -Radiation sterilization
 Non ionizing type, like ultra-violate rays , infra-red rays
 Ionizing type, like Gamma rays , X ray , Beta rays
Ultraviolet Lampe (UV rays)
(300 – 400 nanometers)
Wavelengths 2500-2600 A°
Limited uses that even thin glass or moisture protect from
UV rays
Used in
 sterile inoculations cabinets
 sterile operation rooms
Infra-red sterilize Water bath ….
6
C- GASEOUS STERILIZATION ‫التعقيم بالغازات‬
Equipment:
Special oven for admission of gas, humidity &
hermetic‫محكم السد‬
Gases:
 formaldehyde
 ethylene oxide
 Carbon dioxide
Ethylene Oxide
Kills germs by damaging their DNA-RNA
 Liquid below 11 C
 Flammable, explosive, toxic and possibly carcinogenic
Advantages:
 High penetration
 Compatible with most materials
Disadvantage
 toxic residuals
 Explosive hazard
 Not appropriate for solutions
Application:
 plastic syringes(disposable syringes)
 disposable Petri dish
 surgical sutures
 intraocular lens
 ligament-tendon repair devices‫ إصالح وتر‬- ‫أجهزة الرباط‬
 absorbable bone repair devices
 heart valves and vascular grafts
 Thermolabile powder plastic
 polymers ophthalmic preparation subcutaneous
 vaginal inserts
 tubing sets
7
Mechanical methods:
Filtration:
The material is effect by heat (Heat sensitive solutions )
Ex. (serum , protein , sugar, vaccine,..) are sterilized by filtration .
Filters (bacterial filtration)
1. Porcelain filters
1. Siliceous earth filter
2. Sintered glass filters
3. Asbestos filters
4. Membrane filters  nitrocellulose  different pore
sizes .45μm used
8
D-Others:
 pH,
 Pressure
 Osmotic pressure
 Highly movement speed
 Sonication
Quiz4
Q1- Fill in the blanks with suitable answer:
The disadvantages of gaseous sterilization are __________
and______________.
Q2- Write the types of Wet heat with examples of each type.
9
Chemical methods of sterilization
A- Antiseptic :It is chemical substance that kill micro-organisms on living tissues , ex.
70% alcohol , heptane , 10% Dettol to sterilize hand …
‫المادة الكيمياوية المستخدمة لقتل االحياء المجهرية وتستخدم مع االنسجة مثل االنسان وااليدي‬
B- Disinfectant :IT is a chemical substance used to sterilize non -living objects ‫لتعقيم االشياء‬
‫ الغير حية‬, ex. Phenol , 5% formalin to sterilize refrigerator ‫ الثالجة‬, bench
‫ البنجات‬, floor ‫االرضية‬
The disinfectant may be described either as : Bacteriostatic:- any chemical substance which inhibits the growth
and multiplication of bacteria but do not necessarily kill them .
Bacteriocidal :- any chemical substance which kills the
bacteria
and their spores.
Disinfection : freeing of an object from some or all living pathogenic
microorganism by inhibit growth & multiplication of microorganism .
Sepsis : presence of infection (M.O) in living tissue .
Asepsis : Absence of infection (M.O) in living tissue .
10
Dr.Taghreed Khudhur Mohammad
Ahmad Dawood Salman
Lab.2
/
2015
Preparation of Culture media
Aims:
1. To Isolate the bacteria ‫عزل البكتريا‬.
2. To demonstrate the properties of bacteria ‫ دراسة صفاتها‬.
3. To obtain sufficient pure growth for preparation of antigen and for
other test . ‫زرع نقي‬
4. To determine sensitivity to antibiotic.
5. To estimate viable count . ‫العدد الحي‬
6. To maintain stock culture ( store the microorganisms ffrom months to
years). ‫حفظ البكتريا‬
Equipment:
1. Balance
2. Conical flask.
3. Graduated cylinder.
4.
5.
6.
7.
8.
Spatula.
Source of heat ( Bunsen burner ).
Filter paper
Autoclave
powder of culture media
11
Procedure:1-weight media powder by using a balance.
2-Dissolve powder in D. W
3-use a heat to complete dissolving of powder.
4-put cotton plug on 1 mouth of conical flask.
5-sterilize by using Autoclave . ‫تعقيم االوساط الزرعية باالوتوكليف‬
6-cool the media to (45-50)C .
7-pour this media a Petri dish about 20 ml for each.
8-let plate for some time to Solidify a media.
9-Put plates in refrigerator upside down until using. ‫حفظ االطباق في الثالجة‬
Basic components of culture media:
1- Energy source : (carbon , amino acid ,..)
2- Carbon source : (glucose ,..)
3- Nitrogen source
4- Minerals and salts ( NaCl)
5- pH
6- Growth factor
7- etc….
8- Water
12
Quiz
Q1-Write the advantages of autoclave?
Q2- Why you prepare culture media in your laboratories?
13
Dr.Taghreed Khudhur Mohammad
Ahmad Dawood Salman
Lab.3 /
2015
Classification of culture media according to components
(function) :
1. Simple media
2. Enrichment media
3. Differential media
4. Selective media
5. Special media
Nutrient broth , Nutrient agar
Blood agar , Chocolate agar
MacConkey agar
MacConkey agar, S.S agar, Manitol salt agar
Brucella agar
14
Nutrient agar
25 g
1
L
Distilled water (D.W)
Blood agar :
(N.agar or blood agar) + Distal water = dissolve by heating &then sterilized with
autoclave &then cool to 55c0 &then add 3-4 % blood .
Chocolate agar :
(nutrient agar or blood agar) + Distal water = dissolve by heating &then sterilized with
autoclave &then cool to 80c0 &then add 3-4 % blood (blood is hemolysis release X , V
factor).
15
MacConkey Agar
Is selective for Gram negative organisms, and helps to differentiate
lactose fermenting gram negative rods from Non lactose fermenting gram
negative rods. It is primarily used for detection and isolation of members of
family enterobacteriaceae and Pseudomonas spp.
Composition of MacConeky Agar:
1. Enzymatic Digest of Gelatin, Casein: provides nitrogen, vitamins, minerals
and amino acids essential for growth.
2. Lactose: fermentable carbohydrate providing carbon and energy.
3. Bile Salts: selective agents and inhibit Gram positive organisms.
4. Crystal Violet: Gram positive bacteria are generally inhibited .
5. Sodium Chloride: supplies essential electrolytes for transport and osmotic
balance.
6. Neutral Red: pH indicator. which is red in color at pH’s below 6.8.
When lactose is fermented, the pH of the medium decreases, changing the
color of neutral red to pink
7- Agar : (Solidifying agent ) polysaccharide extract from sea weedy (red algae ) ,
used for solidification of culture media , its solidify at 42 C0 & melted at 95 C 0 .
16g
1 L D.W
16
Classification of media according to solubility (Form) :
1 – liquid media (broth media) :contain all ingredient except agar
(0%).
2 – Semi solid media: contain all ingredient except & 0.2 – 0.4%
agar. (Motility test medium).
+3 – Solid media: contain all ingredient except & 1.5 – 2% agar.
17
Dr.Taghreed Khudhur Mohammad
Ahmad Dawood Salman
Lab.4 /
2015
Inoculation on Solid media (Streaking on Solid media) :
Aim: The purpose of streaking to get an isolated colony to know the type of
Bacteria which is help to diagnose of organism which cause disease.
Equipment :
1.
2.
3.
4.
5.
Spirit lamp or Bensun Burner
Bacteriological loop
Solid media ( Blood agar or Nutrient agar or MacConkey agar , etc. ).
Sample ( urine , stool, blood , CSF,…..etc. ) or Bacteria.
Incubator .
Procedure:
1-Put the solid media and clinical sample near Bunsen burner.
2-Sterilize the loop by flaming .
3- Cool it by touching the loop on side of medium.
4-Hold a piece of colony by loop ( or drop of urine or CSF or stool or
blood or ear swab or wound swab ) and transfer it to a new media .As
in A this area termed inoculum area.
5-Re - sterilize the loop and repeat point 3.
6-make 4 parallel line as in B .
7-Repeat point (2and3) .
8-repeat point 6 as in c
9- repeat point 2.3 .
10- repeat point 6 as in D
11- repeat point 2.3 .
12- from D , Make a zigzag line to the middle of medium to get an
isolated colony .
13- Incubate the culture media in incubator at 37C° for (18-24) hr .
14- Isolated colony appears after incubation.
18
‫ مستعمرة مفصولة‬Isolated colony
19
The method or types of inoculation (culturing)
I-SOLID MEDIA
A-Plate cultured methods
Streak plate method
Carpet culture method
Pouer plate method
B-Tube culture methods
Slope (Slant) culture stock
Deep culture
Stab culture
Roll tubes
II-Liquid media
On SOLID Media
1 -Plate cultured methods (Streak plate method ) :

To isolate single colonies ( Obtaining Isolated
Colonies )
 Colonies can be Identified and Further Evaluated
2-Carpet culture (lawn culture, whole surface, spreading methods).
These methods are prepared by flooding the surface of plate with
suspension of bacteria it provides uniform surface growth of bacteria; it is
useful for bacteria phage typing and antibiotic sensitivity test. And
this method of streaking may be used either for culture from solid
media or for heavy broth culture and smeared over the whole surface,
using a sterile spreader or loop or swabs
20
3-Pouer plate method
About 15-20 ml of agar media are melted and left to cool in water
bath at 45oC-50 C°. Appropriate dilution of inoculum are added in 1 ml
volume to molten agar and mixed well. Contents of tube are poured in Petri
dish. They are allowed to set and after incubation colonies will be seen
distributed through out of the depth medium. The colonies growing in and
on the medium. This method give viable bacteria count in suspension it is
recommended method for food microbiology.
B- TUBE CULTURE METHOD
1- Slope (Slant) culture stock. Many tests devised to
differentiate organisms require solid cultures. It is not always necessary
to grow an organism on a whole Petri dish of medium, and slope
cultures often suffice. ‘Slops’ or ‘slants’ are tubes or bottles containing a
small quantity of medium ,that has been allowed to solidify with the
bottles slightly raised at one end. Such slopes are used only for
maintenance or biochemical tests once the organism has been isolated in
pure culture, they cultured by streaking the surface of slope media
2- Deep culture. Anaerobic organisms require an oxygen-free
atmosphere. For cultivation of these organisms ‘shake’ or ‘deep’ cultures are
sometimes made. The medium is distributed in 150mm x 20mm tubes to a depth of
6-7cm and allowed to solidify. For use, the medium is melted, cooled at about 45 C o,
inoculated with organism, and mixed by rotation between the palms of the hands.
When it has solidified, the culture is incubated and the anaerobic organisms grow at
the bottom of the tube. These shake, or deep, tubes can also be used for counts of
viable organisms.
21
3- Roll tubes. The ‘roll rube’ method is also useful for
counting viable organisms. The medium is distributed in to 6 x
(5/8) in tubes, 1-2 ml per tube, and stored. For use, the medium is
melted, cooled to approximately 50 Co, and a known dilution of the
test sample is added. The tube is then titled and rolled between
finger and thumb, allowing the medium to run all rounds the sides
of the tube just bellow the half way mark. This rolling is carried
out under cold tap water. A thin film of agar solidifies around the
sides of the tube, which is inverted for incubation.
3- Stab culture. It is prepared by puncturing charged long
straight wire (4.5-5 cm). Stab culture are employed mainly for
demonstration of gelatin liquefication and motility test and
for maintaining stock culture
II-Liquid culture
Media prepared in tubes or bottles or flasks and inoculated by touching
with a charged loop. Liquid culture is preferred when large and quick yield
is required. The major disadvantage of Liquid culture is that it does not
provide pure culture from mixed inocula. . This method constitutes a simple
technique and used for liquid culture largely but it may also be used for
culture from solid media.
Quiz / 1 .
Numerate & explain briefly types of culturing.
22
Cultural characteristic
Bacteria grown artificially (in vitro) on agar plates are described as
colonies vary in size, shape, pigment production and hemolysis on blood
agar, depending on the type of media.
A. Description of colonies on solid culture
1- Shape: circular, irregular, radiating or rhizoid.
2- Surface: Bacterial colonies are frequently shiny and smooth in
appearance. Other surface descriptions might be: veined, rough,
dull, wrinkled (or shriveled), glistening.
3- Color – It is important to describe the color or pigment of the
colony. Also include descriptive terms for any other relevant
optical characteristics such as: opaque, cloudy, translucent,
iridescent.
4- Size: Surface colonies are measured in millimeter, they are 2-3
mm in diameter. Smaller ones may be less than (about 0.5-1 mm).
5- Elevation: may be raised, low convex, implicated or dome shape
23
6- Edges: mostly edges are entire, sometimes crenate, fimbrated or
effuse.
7- Color (pigmentation): some organism may produce pigmented
colonies (Staphylococcus,Pseudomonas)
Pseudomonas
Staphylococcus on blood agar
8- Opacity: colonies on nutrient agar may be transparent, translucent
or opaque.
24
9- Consistency: Mostly soft and butyrous and may be hard, firm,
mucoid, tenacious, dry, adherent to medium, friable and
membranous.
Klebsiella on blood agar and MacConkey agar
10-
Contiguity: may be discrete or swarming.
Proteus on blood agar
11- Changes in the medium: colonial growth may bring about color
changes in the media themselves produce soluble pigment that diffuse in
to the medium and some organism haemolysis the blood of medium
around the colony.
25
12Emulsifiability: Growth of some bacteria is easily
emulsifiable (like E. coli, salmonella) where as growth of N.
catarrhalis is not emulsifiable and form granules.
13-
Odor
Description of growth in liquid culture
Growth in liquid medium is described as:
1. Turbidity: Clear or turbid
2. Deposit : Growth of Streptococcus pyogenous is characterized by
deposit at
the bottom of the tube
3. Surface growth: Surface growth is related to aerobic nature of
organism.
4. Color changes: Some organisms produce water soluble, pigment which
after diffusion change the color of medium e.g. Pseudomonas
pyocyneous.
Quiz / 2
How can you describe colonies .
26
Clinical samples :
The samples may be:Blood
Fasces (Stool)
Gastric contents
Hair
Pus
Saliva
Sputum
Urine
C.S.F (cerebrospinal fluid)
Effusions like:-Pleural fluid
Peritoneal fluid
Seminal fluid and others
Swabs like: - Throat Swabs
Pharyngeal Swabs
Laryngeal Swabs
Mouth Swabs
Nasal Swabs
Abacuses Swabs
Ear Swabs
Eye Swabs
Wounds Swabs
Rectal Swabs
Genitourinary swabs:
(High-vaginal Swabs, Urethral discharge
Swabs)
27
Examination of specimens
BACTERIOLOGICAL EXAMINATION OF SPECIMENS
A general plan for examination specimens is as fellows.
1-MACROSCOPIC EXAMINATION:- Note the following:
1. Color, opacity, consistency.
2. Presence of blood, mucus or pus.
3. Presence of macroscopic bodies, such as parasites.
2-MICROSCOPIC EXAMINATION
A- Unstained film or wet preparation
When looking for cells or casts in urine deposit and protozoa
parasites in stool or looking for motile bacteria… ect.
B- Stained film by (a) simple stain
(b) Gram stain
(c) Acid-fast bacilli stain.
(d) Special stains.
(e) Negative staining
3-CULTURE: inoculated accurate media according the suggested
microorganisms
found in the sample, then incubated aerobically and
anaerobically, mostly using
Blood agar.
MacConkey agar.
Special media.
b-Biochemical test:-oxidase, catalase, coagulase , IMVIC tests,
motility,TSI...
c-Serology test and serotyping. agglutination test with specific anti sera
d-Phage typing.
e-Genetic tests.
f- Toxin production.
g-Animal inoculation : is carried out only if necessary.
h- SENSITIVITY TEST Antimicrobial Susceptibility Testing
28
(susceptibility test)
Susceptibility Testing sensitivity of organisms to antibacterial
substances,
e.g.
antibiotics(penicillin,streptomycin,tetracycline,chlorimphenicol,fusidin,
kanamycin,gentamycin,ampicillin,neomycin,furadantin,polymaxins…ec
t.) is an important factor in the treatment of patients. There are two main
methods of sensitivity testing, namely: incorporation methods like discs
Susceptible
Not susceptible
Growth
No
growth
Antibiotic disk
method and diffusion methods. Each of these may be carried out by a
variety of techniques.
Quiz1
Fill in the blanks with suitable answer :There are two main methods for sensitivity test __________
and______________.
29
5- THE REPORT
For the final report, however, it is only necessary to report the
organism or organisms seen in smear and isolated on culture together
with the sensitivity pattern.
30
Microscopic examination of bacteria:
The morphology of bacteria can be studied by the microscopic examination
A- Unstained preparations used to studied both shape and motility of bacteria
suspended in a fluid using:
a -the hanging drop method b- wet smear
B- Staining technique must be used to render
‫للتعرر‬the structure of cells
visible. These will only differentiate relatively gross individual structures
C- Electron microscopy complex techniques are needed to reveal those not
shown by staining.
Staining will help to identify organisms and place them in their own
particular group by their individual reactions to certain stains. An
example is the gram stain.
31
Preparing of bacterial smears
A-Making of wet preparations:
Principle:
drop of liquid containing microorganisms on a slide covered with
a coverslip
Advantages: can observe live organisms
Disadvantages: dries out quickly
Procedure:
1. Make a smear on a clean and dry microscope slide :
a-From culture grow in liquid media by putting 1-3 drops of liquid
cultures on the slide using the loop or Pasteur pipette
b-From culture grow on solid media by emulsify the colony in a small
drop of saline
c-Emulsify the specimens such as feces in a small drop of saline,
iodine, or the required stain.
2-Carefully place a cover slip on to the suspension taking care that no fluid extrudes
beyond the edges of the cover slip.
32
3- Examine microscopically as for hanging
drop under power 10X or
40X.
Hanging drop technique
drop of liquid containing microorganisms
on a slide covered with a cover slip &
suspended over a depression slide
Advantages of the hanging drop:
*Easy to prepare
*Bacteria are live so we can see bacterial
motility:
B-Making of dry smears:
1. Use clean slides free from grease.
2. Mark the slide with a glass writing diamond - grease pencil is easily
rubbed away.
3. From liquid cultures make fairly heavy smears.
4. From cultures on solid media, make thin smears.
5. Do not used water taken form rubber tubing attached to taps for making
smears, as organisms may be transferred from the rubber.
6. When blotting slides, use a fresh portion of paper for each slide, to prevent
33
transference of material.
Bacterial smear
Equipment: Bacteriological Loop.
 Slide (clean and dry).
 Drop of water
(If the culture medium is solid)
 Bunsen burner
 Specimens [growth of microorganisms in
 Liquid media
 Solid media
 Pathological samples ( liquid or solid)
Swab (pus, ear, wound, vaginal ,urethral)
Sputum
C.S.F. (Cerebrospinal fluid)
Urine (after centrifugation)
34
stool,tissue
A//Making of dry smears from liquid media:
Procedure:
1. Sterilize loop in Bunsen flame.
2. Using
aseptic
precautions
‫االحتياطرات‬, with draw one loop full
of culture.
3. Transfer this to a clean slide
4. spread it with the loop to form
a thick film of liquid.
35
5. Sterilize the loop.
4-Allow the film to dry without heating
5-pass the slide 3 times through
the Bunsen film flame.
This
fixes bacteria to the slide.
6-Allow the slide to cool, and then stain the film by the requisite method.
B//Making of dry smears from solid media:
Aseptic precautions must be observed during the manipulation of culture tubes or
plates.
1. Place one drop of distilled water
on
a clean slide by sterilized loop
36
2. Sterilized loop.
3. With the loop transfer to the slide
a small portion of the growth
to be examined and
Emulsify it in the drop of water
until a thin homogenous film is
produced.
4. Sterilize loop in Bunsen flame
5. Allow the smear to dry ( air )
6. Fix by rapidly pass the slide
3 times through the Bunsen film flame.
Fixation

preservation of morphology but NOT internal structures
-Cellular enzymes are inactivated
37
-Cell structures are hardened
kills organisms(Organism dies) [usually results in the death of the attached
microorganisms]
adheres specimen strongly to the
glass slide
promotes stain ability of specimen

Types of fixation:
A//Heat fixing:
1-flame heating bacterial film
Pass slide through flame quickly 3-4 times
Heat fix too little and organisms may wash off slide
Heat fix too much and organisms may be distorted
B//Chemical fixation:
38
*chemical fixatives penetrate cells
*Preserves fine substructures and morphology
*Fixative chemicals penetrate cells and react with proteins and
lipids; (preserves intracellular components) make them inactive, insoluble
and immobile.
1. e.g.:-Acetone, Ethanol, Acetic acid, Mercuric
chloride, Formaldehyde, Glutaraldehyde
Quiz1
Fill in the blanks with suitable answer :1. The advantage of preparing wet smears is to
observe_________.
2. Preparing dry smears is to observe__________.
3. There are two types of fixing bacterial smear
___________and_________.
Note
- Check your answers in key answer page.
39
Staining of smears:
Kinds of bacterial stains
1. Simple stain
2. Compound stain
3. Negative stain
Negative stain
Simple stain
1-Staning around the cell
1-only one dye, one step 2-to
know the morphology of
cells(shape and aggregation)
2-one dye or more
eg:1. Methylene blue (blue)
2. Methyl violet (blue)
3. crystal violet (blue)
4. Natural red (red)
5. Safranin (red)
6. carbol fuchsin (red)
b--- Special stain
40
a---Differential stain
Differential stain
Special stains
1-more than one dye
Mo more than one dye
2-to stain special part of cells eg:-
2-tt to differentiate between two groups
1. Acid fast stain (Z-N stain)
of bacteria
2. Albert's stain
3. spore stain
eg:-Gram's stain:
4. capsule stain
1. Acid fast stain (Z-N stain) ,
5. flagella stain
Ziehl Nelsen stain (tuberculosis).
2. Albert's stain (Corynebacterium)
Simple stain
diphtheria o One reagent
o Usually involves basic dyes
 Crystal violet
 Methylene blue
 Carbol fuchsin
 Most microbes bind basic stains (+ charged dye) because
surfaces have lots of negative charges
 Typical examples are crystal violet and methylene blue)
 Used to stain outer surface, so used to look at morphology, size
and cell arrangement
equipment :-

 Staining rack.
 loop
 Slide.
 Source of heat.
 bacterial growth
simple stain (Crystal violet
or diluted Carbol fuchsin)
 filter paper
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

distil water
70%alcohol
procedure :1- clean and dry slide with
70%70%alcohol
2-prepare bacterial smear
3-fixation the smear
4-put the slide on staining rack.
5- Stain the smear using any simple stain folded the smear by few drops of
stain, let it for 1-2 min.
6-pour the stain from the slide
7-wash the slide by tab water upside.
8-Let it to dry in air or with filter paper.
9-exzmin the stained smear under Microscope by oil (100X)
Differential stain
Gram's stain:
In 1884, Gram described this method which is the most important
stain in routine bacteriology.
It divides bacteria into two categories depending on whether they can
be decolorized with acetone, alcohol, or aniline oil after staining with one
of the rosaniline dyes such as crystal violet, methyl violet, or gentian
violet, and treating with iodine.
Those that resist decolorization remain blue or violet in color and are
designated gram positive, those that are decolorized and take up the red
counterstain such as natural red, safranin or dilute carbol-fuchsin are
termed gram negative.
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Although many investigators have tried to uncover the mechanism of
the gram reaction, no universal answer has yet been found and it is
possible that more than one mechanism exists.
Reagents of gram staim
Solution 1: (primary stain)
Methyl violet
6B (CI No. 42555)………..0.5 g
Distilled water ………………………………………100 ml
Dissolve the methyl violet in distilled water and filter. Record date
and label.
Solution 2 (mordant): Logols iodine
iodine……………..…………10 g
potassium iodide….………….20 g
distilled water………..……….1000 ml
Dissolve the potassium iodide in about 50 ml of water, add the iodine,
dissolve by shaking and make up to the final volume. Record date,
label and store in a tightly stopper bottle.
Solution 3:(Decolorizor)
Absolute ethyl alcohol 95%or Acetone
Solution 4 (Counter stain)
Natural red OR Safranin OR dilute carbol fuchsin
Procedure:
prepare a smear, allow to dry and fix with gentle heat.
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1.
Stain with solution 1 crystal violet (primary stain). for 1-2 min
2.
Treat with Iodine (solution 2) solution (mordant) for 1-2
min. to increase interaction between cell and dye.
3.
rinse with Decolorizer 95% ethanol (solution 3) and continue
application until no more color appears to flow from the
preparation.30sec.
4.
5.
wash with water.
Apply Counter stain ( solution 4 ) safranin, for 1 min. (If dilute
fuchsin is sued, stain for 30 sec.).
6.
Rinse with water. Blot carefully and dry with filter paper.
7.
examine the stained smear under Microscope by oil (100X)
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Gram-positive bacteria remain dark purple
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Gram-negative bacteria pink to red
Table: reaction of some organisms to gram's stain
Gram positive
Gram negative
Staphylococcus
Coliforms
Streptococcus
Neisseria
Pneumococci
Vibrios
Corynebacteria
Spirochetes
Mycobacteria
Salmonella
Bacillus group
Shigella
Hemophylus group
Quiz 3
1. What are the main types of staining with examples?
Steps of stain
Primary stain
Crystal violet
G-ve
Blue
G+ve
Blue
fixation
Lugals Iodine
Blue
Blue
decolonization
Ethanol alcohol
Blue
Colorless
counter Stain
Safranin
Blue
Red
2. What is the procedure of Gram's stain?
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Primary stain
Mordant
decolorization
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5/ Post test:-
Circle the correct answer:1. Hanging drop is used for :a- visualizing live bacteria.
c-a & b
b- Dry smear
c- none
2. Differential stain:a- is used to differentiate between 2 groups of bacteria
is used more tan one dye
cThe
result
will
be
either
G+ve
or
d- all
3. Negative stain :
a- stains around the cell
c- none
b- used one dye or more
d- a & b
4. Heat fixation used to :
a- kill & fix
c-fix only
b- kill the bacteria only
d- none
5. The fixative in Gram's stain is :
a-iodin
b-safranin
c- ethanol
d- decolorizer
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bG-ve
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