Dr.Taghreed Khudhur Mohammad Ahmed Dawood

Lab. 1 2015

Sterilizationالتعقيم Sterilization: is a freeing of an article from all living microorganisms including bacteria and their spores, viruses, yeasts, molds (pathogenic and nonpathogenic)

. البكتريا المتضمنة المجهرية االحياء من االدوات خلو هو ( ممرضة ( والغير الممرضة واالعفان والخمائر والفايروسات وسبوراتها

Methods of Sterilization :- التعقيم طرق

Physical methods .الفيزياوية Chemical methods .الكيمياوية Mechanical methods الميكانيكية

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sterilizationmechanical physical chemical

filtration antiseptic

disinfectant

other

gases

heat

radiation

formaldehyde

pH,osmotic pressure ,highly movement speed ,

sonicationionizing

Non ionizing

Dry heatMoist heatRed heat

Flaming`

incineration

Hot air oven

Temp. above 100 CTemp. at 100 C

Temp below 100 Cautoclaving

tyndalizationpasteurizationboiling

Physical methods of sterilization :- A -Heat sterilization بالحرارة التعقيم

1) Dry heat sterilization اجافة الحرارةa. Red heat used to sterilize wire loops ,point end of forceps

المالقط ونهايات الناقل لتعقيم تستخدم الحمراء الحرارة

b. Flaming: used to sterilize mouth of tubes , glass spreaders

(which are flamed in ethanol ). لتعقيم تستخدم األلهاباالنابيب فوهات

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c. Incineration :الحرق used in pathological fuming

materials .

d. Hot air oven ( 160-180 ) ˚C for 2-4 hr., used to

sterilize glass wares ( pipette زجاجية , ماصات

syringes زجاجية , سرنجات flask زجاجية ,دوارقglass Petri dishes زجاجية بتري فرن. ( etc.… , اطباق

الساخن الحرارة

,Conical flask , volumetric flask , Beaker, Graduated cylinder , Petri dish

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2) Moist heat sterilizationالرطبة a. Temperature below 100˚C, pasteurization (63˚C for 30

min ) , to sterilize milk . من أقل الحليب 100حرارة لتعقيم م

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 .

بدرجة واالوساط 100بخار الجيالتينة االوساط لتعقيم م

السكرية 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 العلميات ومالبس الجراحية االدوات culture media and to sterilize inoculated media الزرعية االوساط Swab المسحة D.W. مقطر , Solutions sealed in containers ampuls, vials , ماء Bulk Solutions كبيرة بحجوم محاليل Glassware Instruments Intraoperative sterilization of metallic devices اجهزة

معدنية metallic surgical instruments

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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 ) Wavelengths 2500-2600 A° (300 – 400 nanometers)

Limited uses that even thin glass or moisture protect from UV rays

Used in sterile inoculations cabinets

sterile operation rooms

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Infra-red sterilize Water bath ….

C- GASEOUS STERILIZATION التعقيم بالغازات

Equipment : Special oven for admission of gas, humidity & hermetic السد محكم

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)

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disposable Petri dish surgical sutures intraocular lens ligament-tendon repair devices - وتر إصالح الرباط أجهزة absorbable bone repair devices heart valves and vascular grafts Thermolabile powder plastic polymers ophthalmic preparation subcutaneous vaginal inserts tubing sets

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 filters1. Siliceous earth filter2. Sintered glass filters3. Asbestos filters4. Membrane filters nitrocellulose different pore

sizes .45μm used

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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.

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Chemical methods of sterilizationA- Antiseptic :-

It is chemical substance that kill micro-organisms on living tissues , ex.

70% alcohol , heptane , 10% Dettol to sterilize hand …

االنسجة مع وتستخدم المجهرية االحياء لقتل المستخدمة الكيمياوية المادةوااليدي االنسان مثل

B- Disinfectant :-

IT is a chemical substance used to sterilize non -living objects لتعقيم

حية الغير الثالجة ex. Phenol , 5% formalin to sterilize refrigerator , االشياء, 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.

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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 .

Dr.Taghreed Khudhur Mohammad Lab.2 / 2015Ahmad Dawood Salman

Preparation of Culture media

Aims:

1. To Isolate the bacteria البكتريا .عزل2. To demonstrate the properties of bacteria صفاتها . دراسة3. To obtain sufficient pure growth for preparation of antigen and for

other test . نقي زرع4. To determine sensitivity to antibiotic.5. To estimate viable count . الحي العدد6. To maintain stock culture ( store the microorganisms ffrom months to

years). البكتريا حفظ

Equipment:

1. Balance 2. Conical flask.

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3. Graduated cylinder. 4. Spatula.5. Source of heat ( Bunsen burner ). 6. Filter paper 7. Autoclave8. powder of culture media

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 . باالوتوكليف الزرعية االوساط تعقيم6-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. في االطباق حفظ الثالجة

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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

Quiz

Q1-Write the advantages of autoclave?

Q2- Why you prepare culture media in your laboratories?

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Dr.Taghreed Khudhur Mohammad Lab.3 / 2015Ahmad Dawood Salman

Classification of culture media according to components (function) :

1. Simple media Nutrient broth , Nutrient agar2. Enrichment media Blood agar , Chocolate agar

3. Differential media MacConkey agar

4. Selective media MacConkey agar, S.S agar, Manitol salt agar

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5. Special media Brucella agar

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 .

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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).

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.

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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 C 0 & melted at 95 C 0 .

16g 1 L D.W

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.

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Dr.Taghreed Khudhur Mohammad Lab.4 / 2015Ahmad Dawood Salman

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 :

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1. Spirit lamp or Bensun Burner 2. Bacteriological loop3. Solid media ( Blood agar or Nutrient agar or MacConkey agar , etc. ).4. Sample ( urine , stool, blood , CSF,…..etc. ) or Bacteria.5. 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.

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The method or types of inoculation (culturing)

I-SOLID MEDIA

A-Plate cultured methodsStreak plate method

Carpet culture method

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Isolated colony مستعمرةمفصولة

Pouer plate method

B-Tube culture methodsSlope (Slant) culture stock

Deep cultureStab 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

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.

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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 Co,

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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.

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.

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Quiz / 1 . Numerate & explain briefly types of culturing.

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

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6- Edges: mostly edges are entire, sometimes crenate, fimbrated or effuse.

7- Color (pigmentation): some organism may produce pigmented colonies (Staphylococcus,Pseudomonas)

Staphylococcus on blood agar Pseudomonas

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8- Opacity: colonies on nutrient agar may be transparent, translucent or opaque.

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

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to the medium and some organism haemolysis the blood of medium around the colony.

12- Emulsifiability: 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 turbid2. Deposit : Growth of Streptococcus pyogenous is characterized by deposit at

the bottom of the tube3. 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.

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Quiz / 2How can you describe colonies .

Clinical samples :

The samples may be:-BloodFasces (Stool)Gastric contentsHairPusSalivaSputumUrineC.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

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Genitourinary swabs: (High-vaginal Swabs, Urethral discharge Swabs)

Examination of specimensBACTERIOLOGICAL 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 EXAMINATIONA- 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 staining3-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

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d-Phage typing.e-Genetic tests.f- Toxin production.g-Animal inoculation : is carried out only if necessary. h- SENSITIVITY TEST Antimicrobial Susceptibility Testing (susceptibility test) Susceptibility Testing sensitivity of organisms to antibacterial substances, e.g. antibiotics(penicillin,streptomycin,tetracycline,chlorimphenicol,fusidin,kanamycin,gentamycin,ampicillin,neomycin,furadantin,polymaxins…ect.) is an important factor in the treatment of patients. There are two main methods of sensitivity testing, namely: incorporation methods like

discs method and diffusion methods. Each of these may be carried out by a variety of techniques.

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Quiz1 Fill in the blanks with suitable answer :-There are two main methods for sensitivity test __________ and______________.

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.

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NNoo ggrroowwtthh

SSuusscceeppttiibbllee NNoott ssuusscceeppttiibbllee

GGrroowwtthh

AAnnttiibbiioottiicc ddiisskk

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 t o render للتعرف the 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.

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Preparing of bacterial smearsA-Making of wet preparations:

Principle : drop of liquid containing microorganisms on a slide covered with a coverslipAdvantages: 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

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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.

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:

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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

transference of material.

Bacterial smear

Equipment:- Bacteriological Loop.

Slide (clean and dry).

Drop of water(If the culture medium is solid)

Bunsen burner

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Specimens [growth of microorganisms in

Liquid media Solid media Pathological samples ( liquid or solid)

Swab (pus, ear, wound, vaginal ,urethral) stool,tissue SputumC.S.F. (Cerebrospinal fluid)Urine (after centrifugation)

A//Making of dry smears from liquid media :

Procedure:

1. Sterilize loop in Bunsen flame.

2. Using aseptic precautions

with draw one loop ,االحتياطات

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full of culture.

3. Transfer this to a clean slide

4. spread it with the loop to form

a thick film of liquid.

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.

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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

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

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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 -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

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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:*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 :-

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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.

Staining of smears:Kinds of bacterial stains

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1. Simple stain 2. Compound stain 3. Negative stain

Simple stain

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b--- Special stain

Simple stain

1-only one dye, one step 2-to know the morphology of cells(shape and aggregation)

eg:-

1. Methylene blue (blue)

2. Methyl violet (blue)

3. crystal violet (blue)

4. Natural red (red)

5. Safranin (red)

Special stains

1-more than one dye

2-to stain special part of cells eg:-

1. Acid fast stain (Z-N stain)

2. Albert's stain

3. spore stain

4. capsule stain

5. flagella stain

a---Differential stain

Differential stain

o more than one dye

2-tt to differentiate between two groups

of bacteria

eg:-Gram's stain:

1. Acid fast stain (Z-N stain) ,

Ziehl Nelsen stain (tuberculosis).

Negative stain

1-Staning around the cell

o One reagento 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 arrangementequipment :-

Staining rack. loop Slide. Source of heat. bacterial growth

simple stain (Crystal violet or diluted Carbol fuchsin) filter paper distil water 70%alcohol

procedure :-1- clean and dry slide with 70%70%alcohol2-prepare bacterial smear 3-fixation the smear4-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.

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Special stains

1-more than one dye

2-to stain special part of cells eg:-

1. Acid fast stain (Z-N stain)

2. Albert's stain

3. spore stain

4. capsule stain

5. flagella stain

Differential stain

o more than one dye

2-tt to differentiate between two groups

of bacteria

eg:-Gram's stain:

1. Acid fast stain (Z-N stain) ,

Ziehl Nelsen stain (tuberculosis).

9-exzmin the stained smear under Microscope by oil (100X)

Differential stainGram'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.

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

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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.

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.

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4. wash with water.

5. 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)

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

Quiz 31. What are the main types of staining with examples?

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Gram positive Gram negative

Staphylococcus

Streptococcus

Pneumococci

Corynebacteria

Mycobacteria

Bacillus group

Coliforms

Neisseria

Vibrios

Spirochetes

Salmonella

Shigella

Hemophylus group

Primary stain

Mordant

2. What is the procedure of Gram's stain?

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Steps of stain G-ve G+vePrimary stain Crystal violet Blue Blue

fixation Lugals Iodine Blue Blue

decolonization Ethanol alcohol Blue Colorless

counter Stain Safranin Blue Red

decolorization

5/ Post test:-

Circle the correct answer:-1. Hanging drop is used for :-

a- visualizing live bacteria. b- Dry smearc-a & b c- none

2. Differential stain:- a- is used to differentiate between 2 groups of bacteria b- is used more tan one dye

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c- The result will be either G+ve or G-ve d- all

3. Negative stain : a- stains around the cell b- used one dye or more c- none d- a & b

4. Heat fixation used to :a- kill & fix b- kill the bacteria onlyc-fix only d- none

5. The fixative in Gram's stain is : a-iodin b-safraninc- ethanol d- decolorizer

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