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Gram Stain Handout

The document outlines the procedure for performing the Gram-stain technique using E. coli and Staphylococcus aureus to study bacterial morphology. It includes a detailed step-by-step guide on preparing heat-fixed smears, staining, and observing the results under oil immersion. Students are instructed to create a flow chart for the procedure and will be assessed on their understanding during the lab session.

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Daniel Evans
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31 views2 pages

Gram Stain Handout

The document outlines the procedure for performing the Gram-stain technique using E. coli and Staphylococcus aureus to study bacterial morphology. It includes a detailed step-by-step guide on preparing heat-fixed smears, staining, and observing the results under oil immersion. Students are instructed to create a flow chart for the procedure and will be assessed on their understanding during the lab session.

Uploaded by

Daniel Evans
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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Gram Stain-Lab VIII

Aim: To learn the Gram-stain technique and to understand its value in the study of bacterial
morphology
Materials:
24 hr culture of E.coli
Staphylococcus aureus
Crystal Violet
Gram’s Iodine
Ethyl Alcohol 95%
Safranin
Slides
Marking pen( sharpie)

Procedure:

Prepare heat-fixed smears of E. coli, S. aureus, and the mixture of E. coli and S. aureus
2. Place the slides on the staining rack.
3. Flood the smears with crystal violet and let stand for 30 seconds
4. Rinse with water for 5 seconds
5. Cover with Gram’s iodine mordant and let stand for 1 minute.
6. Rinse with water for 5 seconds
7. Decolorize with 95% ethanol for 15 to 30 seconds. Do not decolorize too long. Add the
decolorizer drop by drop until the crystal violet fails to wash from the slide. Alternatively, the
smears may be decolorized for 30 to 60 seconds with a mixture of isopropanol-acetone (3:1 v/v).
8. Rinse with water for 5 seconds.
9. Counterstain with safranin for about 60 to 80 seconds (figure 8.3g). Safranin preparations vary
Considerably in strength, and different staining times may be required for each batch of stain.
10. Rinse with water for 5 seconds.
11. Blot dry with bounty tissue paper and examine under oil immersion. Gram-positive
organisms stain blue to purple; gram-negative organisms stain pink to red. There is no need to
place a coverslip on the stained smear. .
Students,

After reading this procedure please make a flow chart so it is easier for you to carry out the
procedure. Do note when you are ready to focus the slide to be viewed you will have to let me
know the name of the organism (Gram +ve or Gram –ve bacteria) and what is the reason behind
your answer (Justification).

Do note that you will be marked for this lab during the lab session so attendance is a must.

Please come early for the lab so we don’t waste time.

Rubric will be posted at a later date.

Thank you

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