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Micro B

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38 views

Micro B

Uploaded by

excessgirl32
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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PREPARATION OF SMEAR FROM AN

AGAR MEDIA

Objective:
 To lay down a procedure for preparation of media for microbiological
tests.
 Scope:
 This SOP is for preparation of media in Microbiology Laboratory.
Materials for smear Preparation:
 culture of Staphylococcus epidermidis
 plate of E. coli
 dye tub
 dye reagents
 clean microscope slides
 wax pencil
 bibulous blotting paper
 immersion oil

Media are of 2 types:


 Solid Media (Agar)
 Liquid Media (Broth)
Method of Preparation of Media:
1. First, make sure that you have a few clean slides. We recycle our microscope
slides and the smears have to be removed from the glass. You will use a
cleansing powder liquid that works well to remove the smear and the dye
from the glass.
 At the sink, pick up the bottle of the slide cleansing liquid, pouring
some of the thick suspension onto the slide.
 Use your finger to spread the suspension over each side of the slide,
and then wash WELL with tap water.
 Dry the slide.
2. You will make smears using 2 different types of cultures, a broth and an agar
culture.
 Label the 2 slides with the names of your bacteria.
 Shaking the culture first, aseptically transfer a drop of the broth culture
using an inoculating loop to a clean slide.
 From the agar plate culture, remove a small inoculum (use only a small
amount of large colonies) with a loop and suspend it in a small drop of
distilled water. Mix the culture well into the water drop.
3. Label 2 slides---Staph and E. coli.
4. Spread the smear suspensions over the glass slide so that it forms a thin layer
and it is the size of nickel or larger no cover slip used
5. Let the slide air-dry—totally. This step facilitates the fixing of the smear to the
slide.
6. Heat-fix the dry smear by running the slide quickly through the flame a few
times. If your fingers get hot, you have heat-fixed TOO MUCH. The heat-
fixing will coagulate some of the protein material and cause the suspension to
adhere to the glass slide.
7. A different dye will be used for each bacterium.
 E. coli and crystal violet
 Staphylococcus and safrinin
8. Staining the smear
 Place the slide on the wire mesh sitting over the dye tub.
 Flood the smear with the dye: let sit for 1 minute.
9. Wash the slide WELL with distilled water. Blot the smear slide with
your bibulous paper pad.
10. Refer to the lab exercise on MICROSCOPY for help with the microscope.
11. Focus on the sample using the 10X lens (Be SURE that you are on brightfield
microscopy).
12. Focus on the smear using the 100X oil immersion lens. Be sure to read the
directions in the MICROSCOPY exercise so that you know how to move to the
100X objective lens using oil.
13. Identify the various shapes and arrangements of bacteria

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