Preperation and fixation of smears for microbiological staining

Introduction

For any microbiological examination, smears of material are most vital. If smears are not properly and fixation is in wrong procedure, exact staining and diagnosis may not be possible.

Labelling of smear

Every slide must be labelled with Patients name, Serial Number, Date and related information necessary for  proper identification

Smear making

smear preparation
smear preparation

Smears should be made an area about 15-20mm,materials evenly spread on side as far as possible. For different type of sample, usually comes in the laboratory for microbiological examination, different technique to be adopted

Purulent smears:

do not centrifuge prulent fluid. Usig a sterile wire loop, make a thin smear.

Non-purulent fluid smears:

centrifuge the fluid, make a smears from well mixed sediment drop.

Culture:

emulsify colony in sterile water & make a thin preparation of the colony. N case of borth culture, tale a loopful to a slide and prepare the smears.

Sputum:

use a clean stick to transfer sputum or purulent materials on a slide. Sterile the stick with phenol or hypochlorite.

Swab:

roll swab on a slide. Rolling the swab avoid breaking of cells, which help to gind intracellular bacteria, like N. Gonorrhoea.

Faeces:

use a clean stick to transfer pus and mucus to a slide. Spread to make a the preparation.

Skin smears:

Necessary for Mycobacterium leprae The specimens are collected by scraping of skin to find out leprae bacilli. It is a superficial invasive procedure, therefore patient counselling is necessary before proceeding.

The procedure are

    1. Wear gloves
    2. Fit scalpel blade to its handle- sterilize with 70% ethanol soaked cotton/ flaming.
    3. Pinch the skin tightly between thumb and index finger so that the area becomes pale and bloodless. Bleeding during procedure hamper finding the bacteria.
    4. Make a small cut about 0.5 cm to the skin with 2-3 mm depth. Continue the skin to hold tightly.
    5. Blot away any blood with dry cotton.
    6. Keep the scalpel blade to the right angle of the cut. Using blunt edge of the blade, scrap firmly 2-3 times along the edge and bottom of the cut to collect a sample of tissue juice and cells.
    7. Transfer the sample to slide, make a small circular smear evenly about 5-7 mm diameter.
    8. Cover the cut with dressing.
    9. Label the slide properly.
    10. Fix with gentle heating keeping smear side upright.

Drying

Dry the smears in air. Protect it from flies, dust, insect and direct sunlight.

In urgent case gentle heating may be done to dry the smears.

Preperation and fixation of smears for microbiological staining
Preperation and fixation of smears for microbiological staining

Fixation

Heat fixation:

widely used procedure, but excess hest damage organism and their staining properties. It also damage WBC, so unsuitable for intracellular organism, like- Gonorrhoeae & N. Meningitidis. However, when used, follow the procedure-

Allow smear to dry completely

Rapidly move slide over spirit lamp or Bunsen burner, keeping the smear upright- no contact with flame. Do it for 2-3 times

Allow to cool before staining.

Alcohol fixation:

less damaging to microorganism and cells. It is the recommended procedure for gram negative intracellular bacteria. Alcohol fixation is more bactericidal than heat. tuberculosis is rapidly killed in sputum smears after applying 70% v/v alcohol.

    1. Allow smear to dry completely
    2. For gram negative intracellular bacteria, fix with 1-2 drops of absolute methanol or ethanol.
    3. For other bacteria, like M. tuberculosis fix with 1-2 drop of 70% v/v methanol or ethanol. (absolute alcohol may be used, but 70% v/v is adequate)
    4. Leave the alcohol for minimum 2 minuets.

Other chemical  fixative

  1. Other chemical are sometimes necessary to fix smears which may contain anthrax bacilli like dangerous bacilli to ensure that bacilli are killed. 40g/l potassium permanganate fixatives are recommended in that case.
  2. Formaldehyde vapour is sometimes used for mycobacterium species. Formaldehyde fixed smears are however poorly stained and formaldehyde vapour are toxic to human.

The smear preparation and fixation is ready and then ready for staining and examination.

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