Preparation & uses of various Staining methods in Microbiology Dr. K.S. Seetha. M.

D Prof & Head Department of Microbiology V.M.K.V.Medical College Salem

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Prerequisites
Film preparations are made on the 3x1 glass slide Slides & coverslips should be perfectly clean & free from grease Commercially available. If not, following procedures must be followed

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Cleaning Slides
For ordinary use: 1. wipe the slide with a clean dry cotton cloth & pass over the flame. 2. Smear the slide with soap solution Remove the film with a clean cloth to make the slide clean & grease-free. For special purpose: Immerse in Conc.Sulphuric acid saturated with potassium dichromate for a day/ more.
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Cover slips
For routine use: Fresh ones are cleaned with clean dry cloth First clean with dichromate solution, wash with tap water, then with distilled water. Store in a stoppered jar in 50% alcohol

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Making Films
In case of fluid material: Urine, pus, sputum. One loopful with inoculation loop spread dry in air fix by heat. In case of solid material; One loopful of water oa slide Transfer a minute quantity of the colony to the drop emulsify spread evenly on the slide dry in air eix by heat.
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Marking the films

Put a circle with a marking pencil on the undersurface of the slide Write the No. or letters on the side end of the slide, before staining

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Staining Methods-1.Simple staining

- Methylene blue, Basic fuchsin - Provide the colour contrast but impart the same colour to all the organisms in a smear Loefflers methylene blue: Sat. solution of M. blue in alcohol - 30ml KoH, 0.01% in water -100ml Dissolve the dye in water, filter. For smear: stain www.similima.com section: stain for 3. For

Simple staining (cont..)

Dilute Carbol fuchsin: - Made by diluting Z-N stain with 10- 15 times its volume of water - Stain for 20-25 seconds, wash with water Use: To demonstrate the morphology of Vibrio cholerae Polychrome methylene blue: Use: MFadyeans reaction - B. anthracis
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2.Negative staining
India Ink, Nigrosin Organisms are not stained, only the background is stained Unstained organisms stand out in contrast Use: To demonstrate the capsule of Cryptococcus neoformans, Streptococcus pneumoniae
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3.Impregnation Method
Bacterial cells and structures that are too thin to be seen under the light microscope, are thickened by impregnation of silver on the surface to make them visible Use: To demonstrate bacterial flagella and spirochaetes
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4. Differential stains
Impart different colours to different bacteria or bacterial structures Eg: Grams stain Acid-fast stain

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5. Grams stain
Originally devised by Christian Gram in 1884 Most widely used stain in bacteriology Differentiates Gram +ve and Gram ve organisms

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Reagents of Grams staining

Crystal violet solution
Crystal violet - 0.5gm Distilled water - 100ml Dissolve in water Lasts longer Does not precipitate To be filtered before use
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Grams iodine
Iodine Potassium iodide Distilled water - 1 gm - 2 gm - 100ml

Dissolve 2 gms of potassium iodide in 25 ml of water and then add 1 gm of Iodine, after dissolving, make up to 100ml
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Decolourizers
Acetone alone - 2 secs Acetone-Alcohol - 10 secs Absolute alcohol - 30 secs

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Counter stains
Safranin Safranin - 0.5 gm Distilled water - 100 ml Dilute Carbol fuchsin Basic fuchsin Distilled water Above soln Distilled water - 0.1 gm - 100 ml - 5 ml - 95 mle3
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Procedure
Prepare the smear, dry in air, fix by heat, stain Cover the smear with crystal violet - 1 min Cover the smear with iodine 1 min Wash with water Cover with decolourizer (alcohol) - 30 secs Wash with water Cover with dil Carbol fuchsin 1 min Wash with water Remove excess water with blotting paper & dry Examine under oil immersion objective.
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Observation

Gm stain photo
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Theories of Gram staining

Acid pH theory Cell wall theory Cytoplasmic theory

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Quality control
A proper staining should clearly differentiate Gm +ve from Gm ve Gm +ve & Gm ve controls to be used Pus cells should always stain Gm ve, can be used as an inbuilt control Always use an unused slide for the preparation of CSF smear Use adequate amount of stain to avoid drying
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Acid-fast staining
Differentiates acid fast and non acid fast organisms Discovered by Ehrlich and modified by Ziehl - Neelsen

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Reagents
Strong Carbol fuchsin - Basic fuchsin - 5gm

- Absolute alcohol - 50ml - 5% Phenol in distilled water 500 ml (475 ml distilled water and 25 ml Phenol) 20% Sulphuric acid - Conc. Sulphuric acid - 20 ml - distilled water - 80 ml Methylene blue (1%) - Methylene blue powder - 1 gm - distilled water www.similima.com - 100 ml

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Procedure
Prepare the smear on a new slide & dry. Fix the smear by gently passing over the flame Flood the slide with strong Carbol fuchsin & until steam rises. Allow the stain to act for 5-7 with intermittent heating. Never too much to produce boiling or charring. The stain must not be allowed to evaporate. Wash with water
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Procedure
Decolourize with 20% Sulphuric acid - 5-7 Wash in running tap water Counter stain with Methylene blue - 1-2 Wash in running tap water Blot dry, observe under oil immersion lens Observation: Acid-fast bacilli --------------------- Pink Tissue &other organisms -------- Blue Result: Smear positive for Acid fast bacilli
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Interpretation of the smear

Grading of the smear: 3-9 bacilli / entire smear -------- 1+ 10 or >bacilli / entire smear------- 2+ 10 or > bacilli/ field ---------- 3+ Report: Smear Positive for AFB ----------1+/2+/3+

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Fluorescent staining ( Auramineo) for Tubercle bacilli

Reagents
Staining solution: Auramine o - 0.3 gm Phenol - 3 gm D.water - 100ml - Dissolve phenol in water with gentle heat - Add Auromine gradually, shake vigorously - Filter & store in a dark stoppered bottle.
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Reagents (cont..)
Decolourizing solution: - Industrial alcohol (ethanol) 75% v/v in water containing 0.5% Nacl & 0.5% Hcl Potassium permanganate solution: - KMno4 soln. - 0.1 gm - Distilled water - 100 ml
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Procedure
Cover the slide with Auramine O soln --- for 15 Wash with water Cover with acid-alcohol ---------------------- for 5 Wash with water Cover with KMno4 soln.----------------------for 30 Wash with water Examine the smear under fluorescent microscope Bright yellow fluorescing bacilli in dark field oil immersion objective
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Alberts staining
Special staining for Corynebacterium diphtheriae To demonstrate the metachromatic granules

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Reagents
A. Staining solution - Toluidine blue - 0.15 gm - malachite green - 0.2 gm - Glacial acetic acid - 1 ml - Alcohol(95% Ethanol) - 2 ml - Distilled water - 100 ml Dissolve the dyes in alcohol and add to water and acetic acid Allow to stand for 1 day and then filter
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Reagents
A. Alberts Iodine - Iodine - Potassium iodide - Distilled water - 2 gms - 3 gms - 300 ml

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Procedure
Prepare the smear, dry in air, fix by heat Cover the slide with Alberts stain for 3 to 5 min Wash with water Cover the slide with Alberts iodine for 1-2 min Wash with water and blot dry Observe under oil immersion objective of light microscope

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Observation
Pale green bacilli Bluish black metachromatic granules

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Photo
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Mycology
KoH Mount: KoH 10gm+ 10ml glycerine+ 80ml D.water Calcofluor white- KoH Preparation: CW 0.05gm+ Evans blue0.02gm+50mlDw - Place a drop of Cw - KoH soln - Add a portion of clinical specimen - Place a coverslip - Observe under L.P & H.P of FM Fungal elements fluoresce blue-white
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Mycology (contd)
India Ink Preparation: - For capsule of Cryptococcus Lacto phenol cotton blue mount: Phenol 20 gm Lactic acid 20 ml Glycerine40 ml Cotton blue 0.05gm Distilled water 20 ml - Add in order. - Used to mount fungi from cultures Grams staining:
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Mycology

Photo
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Parasitology
Demonstration of ova and cysts in stool: Saline mount: for trophozoites and larvae Iodine mount: for cysts Observe under 10 x & 40 x of L. microscope Modified Ziehl - Neelsens stain: For the demonstration of oocysts of Cryptosporidium and Isospora
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Demonstration of blood parasites

Leishman stain Giemsa stain

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Leishmans stain
Stain in powder/ tablet form - 0.15 gm Acetone free pure Methanol - 100 ml
A tablet is ground into paste by adding methanol in small in quantities in a glass mortar The dissolved stain is carefully decanted from time to time in to the glass-stoppered bottle The undissolved stain is ground again with methanol till no residue is left The stoppered glass bottle with the stain is kept in an incubator at 37 c for 24 hours after which it is ready for use
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 Photo of thick and thin smear(page 214) Chatterjee

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Staining of thin film

Pour Leishmans stain on a slide 30 sec Cover the smear with twice diluted stain to prevent drying 10 to 15 min Wash with running tap water and clean the reverse side with wet cotton wool Dry the slide by in an upright position Examine under oil immersion objective Observation: Nucleus red, Cytoplasm blue and RBCs - red
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Staining of thick smear

Dehaemoglobinization Place the slide in a glass cylinder vertically containing distilled water 5 to 10 min When it becomes white take it out and dry in vertical position Stain with Leishmans stain in the same way as that of thin smear
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Giemsa stain
Giemsa powder - 3.8 gms Glycerol - 250 ml Methanol - 250 ml Add methanol to Giemsa powder and dissolve Add Glycerol and place in water bath at 60oc for 3 hours with intermittent shaking

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Procedure
Prepare the smear Fix with pure methanol or ethanol 3 to 5 Diluted stain (5 ml) is added and allowed to dry for 30 to 45 min Wash with running water Dry it in vertical position Observe under oil immersion
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Immunofluorescent staining
Fluorescent dyes - Fluorescent isothiocyanate (blue green) - Lissamine rhodamine (orange red) Fluorescent dyes appear bright under UV light as they convert ultraviolet light into visible light Fluorescent dyes can be conjugated to antibodies and such labeled antibodies can be used to locate and identify antigens
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Direct Immunofluorescence
Used for the identification of bacteria, viruses or other antigens by using specific antiserum labelled with a fluorescent dye. Disadvantage: Separate fluorescent conjugate have to be prepared against each antigen to be tested.

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Direct IF

Photo
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Indirect Immunofluerescence
Eg, Detection of Treponemal antibodies: - Slide with Tr. pallidum as Ag. + - A drop of test serum on a smear - Washed well to remove all free serum leaving behind only Ab.globulin if present on the surface of the Treponemes. - Smear is treated with a fluorescent labelled antiserum to human gammaglobulin
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Indirect IF (contd)
Fluoroscent conjugate reacts with Ab. globulin bound to the treponemes. Wash the slide & examine under UV light Observation: - If Abs.+ in pts serum -----Treponemes appear as bright objects against a dark background. - If Abs in pts serum --- No fluorescence
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Indirect IF
Advantage of the test: A single anti-human globulin fluorescent conjugate can be employed for detecting human Abs to any antigen.

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Indirect IF

Photo
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References
Mackie & Mc Cartney Practical Medical Microbiology, 13th ed Text Book of Microbiology, 7th ed Ananthanarayan & Paniker

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Thank you

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