Lab 8 The Gram Stain

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San Jose State University *

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101

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Biology

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Feb 20, 2024

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1 LABORATORY EXERCISE #8: The Gram Stain Background information Your laboratory instructor will discuss the theory of the gram stain and how it relates to the actual steps of the Gram stain technique. The instructor will also comment on the criteria that must be met to assure a valid Gram stain. A great animation of what occurs to the cell during the Gram stain can be found at: http://faculty.ccbcmd.edu/courses/bio141/labmanua/lab6/images/gram_stain_11.swf Since flash is no longer supported, this can only be played using an app. We will go through this animation on a video recording since many of you will not be able to play it. Adapted from http://www.austincc.edu/microbugz/gram_stain.php : Gram Stain The Gram stain is the most important staining procedure in microbiology. It is used to differentiate between Gram positive organisms and Gram negative organisms. Hence, it is a differential stain. Gram staining involves a four-part process, which includes: ▪ Crystal violet, the primary stain ▪ Iodine, the mordant ▪ A decolorizer made of acetone and alcohol, or just alcohol ▪ Safranin, the counterstain Gram negative and Gram positive organisms are distinguished from each other by differences in their cell walls. These differences affect many aspects of the cell, including the way the cell takes up and retains stains. Gram positive cells take up the crystal violet, which is then fixed in the cell with the iodine mordant. This forms a crystal-violet iodine complex which remains in the cell even after decolorizing. It is thought that this happens because the cell walls of Gram positive organisms include a thick layer of protein-sugar complexes called peptidoglycans. This layer makes up 60-90% of the Gram positive cell wall space. Decolorizing the cell causes this thick cell wall to dehydrate and shrink, which closes the pores in the cell wall and prevents the stain from exiting the cell. At the end of the Gram staining procedure, Gram positive cells will be stained a purplish-blue color.

2 Today's lab activities 1. Perform a Gram stain on your semester unknown (Part A). 2. Review the criteria for a valid Gram stain (Part B). Gram negative cells also take up crystal violet, and the iodine forms a crystal violet- iodine complex in the cells as it did in the Gram positive cells. However, the cell walls of Gram negative organisms do not retain this complex when decolorized. Peptidoglycans are present in the cell walls of Gram negative organisms, but they only comprise 10- 20% of the cell wall space. Gram negative cells also have an outer layer which gram positive organisms do not have; this layer is made up of lipids, polysaccharides, and proteins. Exposing Gram negative cells to the decolorizer dissolves the lipids in the cell walls, which allows the crystal violet-iodine complex to leach out of the cells. This allows the cells to subsequently be stained with safranin. At the end of the gram staining procedure, Gram negative cells will be stained a reddish-pink color. Learning objectives • Explain the basic steps and biochemical reactions of a Gram stain. • Perform and interpret a Gram stain (Gram reaction, cell shape and arrangement). • Identify steps that may result in incorrect Gram reactions of known strains. • Compare and contrast a Gram stain to a simple positive or simple negative stain. Laboratory notebook o Title: Gram stain o Introduction: Some information about the Gram stain ....... The purpose of this lab is to use the Gram stain to determine the cell morphology, cell arrangement and Gram stain reaction of a bacterial unknown. Materials & Methods: Outline your protocol and reference lab manual. o Results: Descriptions and drawings of microscopic fields of Gram stained knowns and unknowns . o Discussion: Interpret your data. What are the cell morphology, cell arrangement and Gram stain of your bacterial unknown? What would you do to improve your Gram stain technique?

3 A. Perform a Gram stain on your semester unknown 1. Review steps for preparing a smear for a simple positive stain. 2. Prepare 3 slides having smears of Staphylococcus aureus , your unknown, and Escherichia coli . Multiple slides are prepared in case the results are poor and new culture preparations need to be stained. This will save you time in the long run. Note: E. coli and S. aureus are commonly used as the Gram-negative (Gm -) and Gram- positive (Gm +) controls, respectively. 3. Air dry and heat-fix all smears. 4. Steps for the Gram stain: a. Flood slide with crystal violet and let stain for 1 minute. b. Drain off crystal violet and briefly rinse with water (< 5 sec). c. Flood slide with Gram's iodine for 1 minute. d. Drain and briefly rinse with water (< 5 sec). e. Remove excess water from the slide so that the alcohol is not diluted in the next step. Decolorize in one of two ways: • Hold the slide on an angle (with a clothespin) and drop 95% ethyl alcohol onto it until the alcohol leaving the slide no longer has a purple tint; be sure to drop the alcohol onto the upper portion of the slide so that the smears are subjected to uniform decolorization. Be careful not to "decolorize" dye from the clothespin! This is the most sensitive and variable step in the procedure and takes experience to know how much to decolorize. Thicker smears require longer decolorization. • Or flood the slide with 95% ethyl alcohol. Leave for 10-30 seconds, then rinse with distilled water.

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4 f. Drain the slide then flood the slide with safranin and let stain for 2 minutes. g. Rinse with distilled water and blot dry with bibulous paper. 5. Examine the smears with the oil immersion objective; ALWAYS EXAMINE THE CONTROL ORGANISMS FIRST to insure the stain has been done properly. Record your results which will go into your lab write-up. A summary statement of your results (e.g., Gm + cocci in cluster or Gm - rods) should also be recorded under exercise in your unknown report worksheet. B. Review the criteria for a valid Gram stain 1. Thin smear. Why? 2. Young culture (18-24 hours). Why? 3. Proper reagents. 4. Use of appropriate controls. C. Clean up: When you have finished: • TURN OFF YOUR FLAME. • Return Unknown slants to the fridge. • Follow instructions in Lab 1 to put away your microscope properly. Make sure your instructor checks that your microscope is clean before you put it away. • Discard slides and cover slips in the DC Gold container . DO NOT put gloves or paper in the DC Gold. • Toss used bibulous and lens paper in the normal trash. • Empty stain waste from the tray into the Stain Discard jug. Do this frequently if your tray is filling up. Use the funnel to transfer the liquid. Rinse the stain tray with DI water into the Stain Discard jug until it runs clear. Replace the cap of the Stain Discard jug when done. If the jug is filling up, get a new jug from the bottom shelf of the Discard cart. • Wipe down labels on in-class Gram stain slants with 95% ethanol. Place tubes in a rack on the Discard cart. • Return stain bottles to their secondary containers on the back sink. • Return strikers, lens cleaner, and immersion oil to the front bench. • Wipe down your bench with 70% ethanol: Spray surface with ethanol, spread with a Kim wipe, and allow to air-dry. Toss Kim wipe in the normal trash . Return ethanol spray bottle to its secondary container in the back of the sink. • Toss gloves in the large red biohazard bin . • Follow the general lab clean-up instructions to exit the lab.

5 D. Pre-lab Questions: 1. How does a differential stain differ from a simple stain? Is a Gram stain simple or differential? 2. What procedure needs to be completed before you can start adding your stains? 3. Fill in the diagram: E. Questions for Lab Write-up #1: 1. Outline or discuss what happens at the cellular level to a Gram negative and Gram positive cell at each step during the Gram staining procedure. 2. Why is it important to make a Gram positive and a Gram negative control on the same slide as your Unknown microbe? 3. What does it mean to have a Gram variable sample, and why can Gram variability occur? References: 1. Salton, J. R. J. 1961. The Anatomy of the Bacterial Surface. Bacteriological Reviews, 25:77-99. 2. http://www.austincc.edu/microbugz/gram_stain.php

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