lab report

Topic: Gram Stain

The written report will include the following components:

· Introduction – Introduce the purpose of the experiment and your results/methods. Explain any ideas or techniques that are necessary for someone to understand the results section. (1-3 page) 20 points

· Results – Include ONLY relevant figures and tables. 35 points

· Discussion – Summarize what you have found and explain the importance or implication. (1 page) 35 points

· References – Follow reference formatting given in the (no page limit). Use as least 2 alternative sources as a reference. 10 points

· Other relevant information (optional)

Presentation guidelines:

Time limit: 10-15 minutes

The purpose is to share the results of your experiment in class.

Due to time constraints, limit your presentation to ca. 7 slides

Gram Stain

Abstract

Introduction

Many different stains and staining procedures are used in Microbiology, involving more than one chemical stain to differentiate between distinct microorganisms or cellular components of a single organism. Gram stain, as called Gram’s method, is one method of staining to distinguish whether bacterial species are Gram positive or Gram negative.

The gram staining technique was named after a Danish bacteriologist, Hans Christian Gram, who devised the method in 1884 to contrast tissue sections for observing. The technique later plays a major role in classifying bacteria and continues to be a standard procedure in medical microbiology.

It was found that Gram staining differentiates bacteria by different properties, both physical and chemical, of the cell walls. Gram-positive bacteria have thick cell walls of peptidoglycan, retaining the primary stain of crystal violet, while Gram-negative bacteria have much thinner walls of peptidoglycan surrounded by an additional lipid membrane, allowing the crystal violet to be washed out and showing pink or red by counterstain.

In the process of Gram stain, the bacterial smear is air-dried and then heat-fixed to adhere to the glass slide, followed by staining. Crystal violet is usually the primary stain in this method but can sometimes be substituted by equally effective methylene blue. The bacterial cell wall is first stained by crystal violet, and then iodine solution is added, acting as a mordant in the form of crystal violet-iodine complex to bind the dye to the cell. This step of gram staining is commonly referred to as fixing the dye.

During the next step of decolorization, 75% alcoholic solution or a mixture of ethanol and acetone is used to dissolve the lipid layer of the gram-negative cells, leaching the primary stain on the cells into the surrounding solvents. And then the smear is counterstained with safarin. Gram-positive organisms that have the purple crystal dye complexed with the iodine solution remain to be purple as the dye is not removed by the alcohol. On the other hand, the Gram-negative organisms have had the purple stain removed by the alcohol and the colorless cells take up the red color of the safranin counterstain.

Material

· Safety Gear

· Goggles

· Gloves

· Lab coat

· Face shield

· Staining Kit

· Crystal violet, a few drops

· Gram’s iodine, a few drops

· Ethyl Alcohol 95%, a few drops

· Safranin O solution, a few drops

· Lab equipment

· Microscope

· Forceps

· Glass slide, 1

· Inoculating loop, 1

· LB agar plate with colonies from lab 1.3

· Bunsen burner

· Striker

· Marking pen

· Clean-up

· 500 mL clean beaker

· DI water

· Ethanol wash bottle

· Hand sanitizer

· Disposable pipettes (if needed)

· Paper towel

· Lens paper

· Kimwipes

Procedure

1. Sanitize hands and then put on safety gears.

2. Sterilize the workbench with ethanol and wipe clean some paper towels.

3. Disinfect gloved hands with ethanol again and wait until they are dry.

4. Mark a circle on the slide with a marker on the bottom side of the slide.

5. Put some DI water in a beaker.

6. Set up and light the bunsen burner.

7. Sterilize the inoculation loop with the flame, starting from the base, until the loop is red hot.

8. Use the loop to transfer a drop of DI water from the beaker to the slide. Use as little as possible.

9. Place the drop within the marked circle.

10. Sterilize the loop again, as described in step 7.

11. Let the loop cool near the flame.

12. Lightly touch a colony from the streaking culture from the previous lab to obtain a small amount of bacteria.

13. Mix the bacteria with the drop of DI water on the slide within the circle until the mixture becomes slightly cloudy.

14. Repeat step 7 to sterilize the loop.

15. Rest the loop on the side of the burner to let cool.

16. Let the slide dry in the air without moving it for about 5 minutes.

17. Use forceps to pass the slide through the flame 2 to 3 times to heat fix the organisms to the slide.

18. Turn off the burner.

19. Put the slide on a waste beaker.

20. Drop a few drops of crystal violet until it covers the circle.

21. Leave the crystal violet on the slide for ONLY 60 seconds, then immediately raise with DI water over the waste beaker.

22. Gently blot the remaining water on the slide with some kimwipes. DO NOT rub from side to side.

23. Drop a few drops of Gram’s iodine until it covers the circle.

24. Leave the Gram’s iodine on the slide for 30 seconds, then wash it off with DI water over the waste beaker.

25. Repeat step 22 to remove any water left.

26. Hold the slide at an angle, about 45 degrees, and decolorize the slide over the waste beaker with 99% ethanol until no more purple dye runs off, usually ONLY about 20 seconds.

27. After decolorization, raise the slide with DI water over the waste beaker and repeat step 22 to remove excess water.

28. Add safranin to the slide and leave it on for about 30 seconds.

29. Wash the slide with DI water over the waste beaker and repeat step 22 to remove excess water.

30. Wait until the slide is completely dry.

31. Mark a larger circle on the upper side of the slide and wipe off the original one with ethanol.

32. Observe the slide under the microscope.

33. Use the marked circle as a positive control starting at 10x.

34. Once have a sharp image, move the slide to find the stain.

35. Use the fine adjustment to find a sharp image at 10x.

36. Rotate the lens to 20x and repeat step 35.

37. Rotate the lens to 40x and repeat step 35.

38. Record each image by taking a picture and share with other group members later.

Result

4x, 10x, 20x, 40x

Discussion

Reference