Path19-Staing & All

8/3/2019 Path19-Staing & All

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Preparation & uses of various

Staining methods in Microbiology

Dr. K.S. Seetha. M.D

Prof & Head

Department of Microbiology

V.M.K.V.Medical CollegeSalem

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Prerequisites

Film preparations are made on the 3x1

glass slide

Slides & coverslips should be perfectlyclean & 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 solutionRemove 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 asmear

Loefflers methylene blue:

Sat. solution of M. blue in alcohol - 30mlKoH, 0.01% in water -100mlDissolve the dye in water, filter.

For smear: stain for 3. For section: stain

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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 - 0.5gm

Distilled water - 100ml

Dissolve in water

Lasts longer

Does not precipitate To be filtered before use

Crystal violet solution

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Grams iodine

Iodine - 1 gm

Potassium iodide - 2 gm

Distilled water - 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 secsAbsolute alcohol - 30 secs

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Counter stains

SafraninSafranin - 0.5 gmDistilled water - 100 ml

Dilute Carbol fuchsin

Basic fuchsin - 0.1 gm

Distilled water - 100 mlAbove soln - 5 mlDistilled water - 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.17www.similima.com


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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 clearlydifferentiate Gm +ve from Gm ve

Gm +ve & Gm ve controls to be used

Pus cells should always stain Gm ve, canbe 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 mldistilled water - 80 ml

Methylene blue (1%)- Methylene blue powder - 1 gmdistilled water - 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 --------------------- PinkTissue &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 gmPhenol - 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 towater and acetic acid

Allow to stand for 1 day and then filter

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Reagents

A. Alberts Iodine

- Iodine - 2 gms

- Potassium iodide - 3 gms

- Distilled water - 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 lightmicroscope

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Observation

Pale green bacilli

Bluish black metachromatic granules

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Photo34www.similima.com


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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 mlGlycerine40 mlCotton blue 0.05gmDistilled water 20 ml- Add in order.- Used to mount fungi from cultures

Grams staining:36www.similima.com


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Mycology

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Parasitology

Demonstration of ova and cysts in stool:

Saline mount: for trophozoites and larvae

Iodine mount: for cystsObserve 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

A tablet is ground into paste by addingmethanol in small in quantities in a glass mortar

The dissolved stain is carefully decantedfrom time to time in to the glass-stoppered bottle

The undissolved stain is ground again with

methanol till no residue is leftThe stoppered glass bottle with the stain is kept

in an incubator at 37 c for 24 hours after which itis ready for use

Stain in powder/ tablet form - 0.15 gm Acetone free pure Methanol - 100 ml

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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 toprevent drying 10 to 15 min

Wash with running tap water and clean thereverse 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

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 sameway as that of thin smear

Dehaemoglobinization

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Giemsa stain

Add methanol to Giemsa powder anddissolve

Add Glycerol and place in water bath at 60oc

for 3 hours with intermittent shaking

Giemsa powder - 3.8 gmsGlycerol - 250 ml

Methanol - 250 ml

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Procedure

Prepare the smear

Fix with pure methanol or ethanol 3 to 5

Diluted stain (5 ml) is added and allowedto 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 UVlight as they convert ultraviolet light intovisible light

Fluorescent dyes can be conjugated toantibodies and such labeled antibodiescan 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 labelledantiserum 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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