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LAB WEEK 4 Gram Staining

The document provides instructions for performing a Gram stain, which is used to differentiate between gram-positive and gram-negative bacteria under a microscope. It describes cleaning slides, making a bacterial smear, heat fixing, staining with crystal violet, iodine, decolorizer, and safranin, and viewing under a microscope. Several key differences when performing the technique in an actual laboratory are also outlined, such as wearing protective equipment and maintaining sterile technique to avoid contamination.

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35 views4 pages

LAB WEEK 4 Gram Staining

The document provides instructions for performing a Gram stain, which is used to differentiate between gram-positive and gram-negative bacteria under a microscope. It describes cleaning slides, making a bacterial smear, heat fixing, staining with crystal violet, iodine, decolorizer, and safranin, and viewing under a microscope. Several key differences when performing the technique in an actual laboratory are also outlined, such as wearing protective equipment and maintaining sterile technique to avoid contamination.

Uploaded by

tonistack847
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
You are on page 1/ 4

ID: __________________________

Materials Required:

1. Clean glass slides


2. Inoculating loop
3. Bunsen burner
4. Bibulous paper
5. Microscope
6. Immersion oil
7. Distilled water

Reagents:

1. Primary Stain - Crystal Violet


2. Mordant - Grams Iodine
3. Decolourizer - Ethyl Alcohol
4. Secondary Stain - Safranin

Procedure:

Part 1: Preparation of the glass microscopic slide

Grease or oil free slides are essential for the preparation of microbial smears. Grease or oil from
the fingers on the slides is removed by washing the slides with soap and water. Wipe the slides
with spirit or alcohol. After cleaning, dry the slides and place them on laboratory towels until
ready for use.

Part 2: Labeling of the slides

Drawing a circle on the underside of the slide using a glassware-marking pen may be helpful to
clearly designate the area in which you will prepare the smear. You may also label the slide with
the initials of the name of the organism on the edge of the slide. Care should be taken that the
label should not be in contact with the staining reagents.

Part 3: Preparation of the smear

 Bacterial plate cultures: With a sterile cooled loop, place a drop of sterile water or
saline solution on the slide. Sterilize and cool the loop again and pick up a very small
sample of a bacterial colony and gently stir into the drop of water/saline on the slide to
create an emulsion.

Please note: It is very important to prevent preparing thick, dense smears which contain an
excess of the bacterial sample. A very thick smear diminishes the amount of light that can pass
through, thus making it difficult to visualize the morphology of single cells. Smears typically
require only a small amount of bacterial culture. An effective smear appears as a thin whitish
layer or film after heat-fixing.

Part 4: Heat Fixing


ID: __________________________

Heat fixing kills the bacteria in the smear, firmly adheres the smear to the slide, and allows the
sample to more readily take up stains.

 Allow the smear to air dry.


 After the smear has air-dried, hold the slide at one end and pass the entire slide through
the flame of a Bunsen burner two to three times with the smear-side up.

Now the smear is ready to be stained.

Please Note: Take care to prevent overheating the slide because proteins in the specimen can
coagulate causing cellular morphology to appear distorted.
Part 5: Gram Stain Procedure

1. Place slide with heat fixed smear on staining tray.

2. Gently flood smear with crystal violet and let stand for 1 minute.

3. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash
bottle.

4. Gently flood the smear with Gram’s iodine and let stand for 1 minute.

5. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash
bottle. The smear will appear as a purple circle on the slide.

6. Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the
alcohol drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful
not to over-decolorize.

7. Immediately rinse with water.

8. Gently flood with safranin to counter-stain and let stand for 45 seconds.

9. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash
bottle.

10. Blot dry the slide with bibulous paper.

11. View the smear using a light-microscope under oil-immersion.

Differences Encountered in a Real laboratory:

1. Always wear gloves, and lab coat.


2. Tie your hair properly to prevent any contamination from the culture you are working
with.
3. After entering the lab, make sure that the microscope is working properly. Oculars and
objective lenses should be cleaned before and after each use with lens paper.
ID: __________________________

4. Adjust the illumination before using the microscope.


5. Prepare your work space (Laminar Air Flow Cabinet) or lab bench by wiping down the
area with disinfectant.
6. Properly adjust the flame of the Bunsen burner. The proper flame is a small blue cone; it
is not a large plume, nor is it orange.
7. Wipe the glass slide with spirit and wave the slide over the Bunsen burner to remove any
unwanted microorganisms in the slide.
8. While flaming the inoculation loop be sure that each segment of metal glows orange/red-
hot before you move the next segment into the flame.
9. Once you have flamed your loop, do not lay it down, blow on it, touch it with your
fingers, or touch it to any surface other than your inoculums. If you do touch the tip to
another surface or blow on it, you will have to re-flame the loop before you proceed to
your experiment.
10. Allow your loop to cool before you try to pick up your organism. If you pick up
organism with a hot inoculation loop, your cells will be killed and will affect your results.
11. When removing the caps from tubes, always keep the caps in your hand. Never set them
on the table, as they could pick up contaminants.
12. Always handle open tubes at an angle near to the flame of the burner; never let them
point directly up, since airborne or other environmental organisms could fall into the tube
and cause contamination.
13. As soon as you transfer the organism into the slide, flame your loop. Never place a
contaminated tool on your workbench.
14. Try to prepare a single cell layer of organism (a thin smear). Otherwise the all cells will
appear as gram positive in thick area.
15. Do not overwarm the cells. Dry the slide thoroughly prior to heat fixing.
16. Use young, vigorous cultures rather than older cultures for your experiment.
17. Decolorisation step should not exceed the time limit.
18. While washing the slide after staining, do not let the water stream fall directly on the
smear. This may disrupt the smear. Let the stream of water flow slowly along the surface,
such that only the stain is flooded and the smear is intact.
19. Always prefer to observe under 10X first. This will give you an idea of the location of a
good area for observation. After this you may prefer to switch over to 40X.
20. Do not ever observe at a specimen at 100X without oil.
21. While focusing the microscope, glass slides should be handled carefully to avoid the
chance of chipping or breaking.
22. After the observation, wipe the microscopic lens with an absorbent paper and cover the
microscope properly.
23. Discard all contaminated materials properly and return your supplies to the proper storage
locations, and clean up your working area.
24. Always disinfect your work area when you are finished.
25. Ensure proper hand washing before you leave from the laboratory.
ID: __________________________

Questions

1. Why is it important to follow sterile technique?


______________________________________________________________________________
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2. What can happen to cause gram-positive bacteria to appear gram-negative?
______________________________________________________________________________
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3. Name three gram-positive bacteria and the diseases they cause.

______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

4. Name three gram-negative bacteria and the diseases they cause.

______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

5. Which step in the Gram stain procedure is most critical?

______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

6. What two cell characteristics can be determined from using the Gram stain?

______________________________________________________________________________
______________________________________________________________________________

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