Gram Stain

7. Gram Stain and KOH Test Introduction Gram stain procedure is the most used staining procedure in pathology labs. It is fast, inexpensive and informative; however, to exclude the possibility of false positive and false negative results, a number of precautions must be taken. Old cultures should not be used because degraded cell walls in dying Gram (+) cells give a false negative result. Thick smears, over- or undercolorization will give false results. A known sample is usually included as a control of the staining procedure.

Gram stain is use to stain bacteria as a first step for their identification. Gram stain differentiates between two types of cell wall. Bacteria with cell walls containing a large layer of peptidoglycan and no lipopolysaccharide are Gram positive. Bacteria with cell walls containing a thin layer of peptidoglycans and an outer cell wall containing lipopolysaccharides are Gram negative. Mycobacteria, e.g. the causative agent of tuberculosis, and Nocardia, do not stain well with Gram stain and require a special stain called acid fast.

The exact mechanism of the Gram stain is not fully understood and does not seem to be directly connected to the actual structure of the cell wall. This vexing fact does not detract from the usefulness of the staining procedure itself!

Here is a great site that summarizes the technique and use of Gram stain in pathology.

An easy technique (Ryu 1938) also characterizes the Gram stain reaction to a large extent. We use this test in our lab to complement the results of the Gram test. It is not a replacement for the commonly used Gram stain. We will test the same bacteria that you stained in the preceding exercise: Escherichia coli, Staphylococcus epidermidis, and Bacillus megaterium. Neisseria sicca is an example of a Gram negative coccus and was not used in the Simple Stains. We do not use Candida in this exercise.

Relationship to Class Instruction: Chapters 3 and 10.

Lab report due on: _______________________________________

Gram stain of Staphylococcus epidermidis and Escherichia coli. 1,000x

7-1

Goal of Laboratory 

Perform a successful Gram stain



Use of KOH as an Indicator of Gram Stain Reaction

 All procedures and results are entered in the lab notebook and initialized by instructor before leaving class

Materials and Methods Organisms Bacillus megaterium: rod-shaped bacteria, arranged in strands Escherichia coli: bacilli, rod-shaped bacteria, scattered Staphylococcus epidermidis: cocci, round shaped bacteria, arranged in clusters Neisseria sicca: round shaped bacteria, notice kidney shape and diplococcic arrangement, known to decolorize poorly Clothespins

Gram stain reagents

Microscope slides

1. Crystal Violet

Sterile toothpicks

2. Iodine solution

Heating plates

3. Decolorizer (alcohol/acetone) 4. Safranin 3% KOH Distilled water droppers

Procedure Special Safety Precautions Discard all biohazard material appropriately: Slides go in the metal tray.

Smear Preparations: see Simple Stains.

Each pair of students will prepare four slides: two slides by each student. Choose either E.coli and B. megaterium or N. sicca and S epidermidis.

Pointers for a successful Gram stain A clean slide A good bacterial smear Don’t use too much material. It will be difficult to decolorize reliably and distinguish morphology. The smear should be just cloudy.

Let it air dry first.

Remember to fix with heat by passing the slide, sample side up, through the flame. Do not overdo this step otherwise you will destroy the morphology of the microorganisms.

7-2

Add the decolorizer drop by drop to control the process. It does not take long unless the sample is too thick. Do not squirt water from the wash bottle. It is a gentle wash.

Gram Stain the process by placing the slide on a warm plate.

1. Place the slide on paper towel, smear side up.

If more than one slide is placed on the paper towel at one time, do not allow the slides to touch each other.

11. Examine the stained slides with low power objective first.

2. Add 2-3 drops of crystal violet stain directly

12. For this you will need maximum light by

on the smear. Stain for one minute.

opening the diaphragm and adjusting the light source intensity.

3. Rinse the slide by washing the stain off with a gentle jet of water from a wash bottle (at this stage all bacteria will be stained purple by the crystal violet).

13. Examine the bacteria and observe the size, shape (rod or spherical), Gram staining (positive: purple; negative: pink), and arrangement (singly, in pairs, in chains, irregular clusters, or regular packets of four or eight). Compare the Gram negative organism(s) with the Gram positive and note the difference in color. If not clearly evident, check your procedure with the instructor.

4. Drain off the rinse water.

5. Add 2-3 drops of Gram's iodine solution. Let the slide stand for one minute. The mixture will turn dark purple and look almost black.

6. Rinse with water as described in step 3 above.

NOTE: Achieving a good focus with oil immersion is frequently difficult and time-consuming; be patient and get help!

7. Decolorize the stain by adding the decolorizer mixture drop by drop. Let it run down over the slide, which should be held at an angle, until the stain is no longer being removed from the slide. Quickly rinse the slide with water.

Remember oil immersion procedure. Only the oil immersion objective is used with immersion oil. Do not use other objectives once you used oil not to dirty the other objectives. When you are done, wipe the objective clean with lens paper. (ASK INSTRUCTOR, if you cannot find any.)

NOTE: A thick stain takes longer to decolorize than a thin one, so the exact time cannot be specified. It is usually not more than 20 seconds. Too little or too much decolorization can affect your results. At this stage the Gram positive organisms will remain purple and Gram negative bacteria will be colorless. A thick smear will be clearly visible. A thin smear from a broth culture may not be visible at all at this stage.

14. Sketch the organisms seen on the slides on the last page of the instructions. Enlarge your drawing enough so that the arrangement, shape and relative size of the organisms can be shown. Do not try to sketch the entire field. Show the instructor the slide for initials on records. You will include the carbon copies to your lab report.

I will initialize your results.

8. Add 2-3 drops of safranin stain and let stain for 30 seconds.

9. Rinse the slide with water as described in step 3 above.

10. Air-dry the slide. The slide should be completely dry before adding oil for microscopic examination. You can speed up

7-3

KOH Test 3. Raise and lower the toothpick after 5-8

1. Place one small drop of 3% potassium

seconds, to determine the stringing effect. If there is stringing (increased viscosity) within 15 seconds, the bacteria are considered to be Gram negative. Do your results from this test agree with those from the Gram stain? Record your observations.

hydroxide (KOH) solution on a clean slide.

Test E coli, B megaterium, S epidermidis, and N sicca. Transfer a good amount of bacteria with a sterile toothpick from the culture medium to the drop.

2. Mix the bacteria into the solution rapidly with a circular motion.

Klebsiella pneumoniae Photo courtesy of Wayne Curtice, Lake Washington Institute of Technology

Lab Report Introduction: Goal of experiment and reason to perform the experiment.

Materials and Methods: give a brief description of what you did. Refer reader to “Microbiology Manual lab 5” for details. Don’t forget to record which organisms were stained. Use scientific style for the names of bacterial cultures: Escherichia coli or Escherichia coli. Abbreviate the genus after the first use: e.g.

E coli.

Results: Describe in words cell morphology (spheres, rods) and arrangements (group, single cells).

Describe cell morphology and color reaction. Include results from the KOH test. Carbon copies must be stapled to the lab report.

Discussion: Compare the shape and arrangement of the different cultures. You stained only one or two bacterial cultures, but you also observed the slides of your lab partners. Compare simple stains and Gram stain. Did the results match what was expected? If the staining results are different than what is reported in the literature, what could have happened? Did you notice endospores in the Bacillus samples?

Compare the results you obtained with the KOH test to the results of the Gram stain.

Conclusion: General comments about the whole exercise. How did the lab add to your knowledge of microbiology? How would you improve your results, if they needed improvements? How is Gram stain used in pathology labs? Why is the Gram stain still so important? Do all bacteria classified as Gram positive always stain Gram positive? See textbook and class notes.

Staple the page with drawings adding the final magnification to all observations and carbon copies of lab notebook.

For more details on how to write a lab report, refer to “How to write a lab report” in the lab manual.

7-4

Lab 7. Gram Stain of Bacteria Bacillus megaterium

Escherichia coli

Neisseria sicca

Staphylococcus epidermidis

Signature of instructor:_____________________________

7-5