ENVE 301: Environmental Microbiology Laboratory

Differential Staining:

The Gram Stain

The only faith that wears well and holds its color in all weathers, is that which is woven of conviction and set with the sharp mordant of experience.

- My Study Windows. Abraham Lincoln - James Russell Lowell, 1864.

Supplemetal Reading/Web Sites

Pink and Purple Bugs:

The Gram Stain Technique

How to do Gram Stains

Simple staining of bacterial cells (See lab 2) does not provide much information to aid in the identification of bacterial cells. In addition, as early medical microbiologists discovered, all cells, both prokaryotic and eukaryotic will stain with the dyes used in simple staining. The Gram staining method, named after the Danish bacteriologist who originally devised it, Hans Christian Gram, is one of the most important staining techniques in microbiology. In 1883-4 Dr. Gram discovered a method for staining certain bacterial cells purple/blue while staining eukaryotic cells, and many other bacterial cells pink. Essentially, Dr. Gram found that bacteria fell into two distinct categories when stained sequentially with crystal violet followed in sequence by a bath in an iodine solution, a wash with a destaining agent and a counter-stain. One group of cells resisted the removal of the crystal violet when washed with ethanol or acetone (decolorizing agents), whereas the second group was readily decolorized by a brief rinse with these reagents. To visualize the decolorized cells Gram briefly exposed them to a counter-stain, or a stain of a different color from the crystal violet. Gram settled on the red counter stainingdye safranin. Thus cells which resisted decolorization remained deep purple or blue and came to be referred to a Gram positive cells, whereas cells that easily lost the crystal violet dye were red after counter staining. These red cells came to be called Gram negative cells.

This type of stain is referred to as a differential stain because one group of organisms reacts to the stain one way and another group of organisms reacts a different way. Gram and others drew several important conclusions from these staining results. First, they realized that differential staining of cells and cell components was possible. Secondly, they recognized that this staining was diagnostic and could be used to identify cells and substances. We now know that Gram positive bacterial cells are very different from Gram negative cells. These differences have proven useful in understanding the physiology of these two categories of bacteria. The gram characteristic is almost as fundamental to a bacterial description as its morphology.

The ability of the Gram stain to differentiate microorganisms into two broad categories is a result of differences in the cell walls of the organisms. The cell walls for gram-negative microorganisms have a higher lipid content than gram-positive cells. Originally, both kinds of cells are penetrated by the crystal violet. Iodine is subsequently added as a mordant to form the crystal violet-iodine complex so that the dye cannot be removed too easily. This step is commonly referred to as fixing the dye. The actual mechanism of decolorization is currently not well understood and remains controversial. However, it is generally agreed that the subsequent treatment with the decolorizer, which is a mixed solvent of ethanol and acetone, dissolves the lipid layer from the gram-negative cells. The removal of the lipid layer enhances the leaching of the primary stain from the cells into the surrounding solvent. In contrast, the solvent dehydrates the thicker gram-positive cell wall, closing the pores as the cell wall shrinks during dehydration. As a result, the the diffusion of the stain-iodine complex out from the cell is obstructed, and the cells remain stained.

Performing a good gram stain is easy, but it does require some experience. For example, the original "bacterial smear" must not be too thick or too thin as this will influence penetration of the staining reagents.

Also the age of the culture is important. Very young and very old cells often produce poor results, whereas mid-log cells that are healthy and growing optimally, tend to give dependable results. The media the cells are grown in and the environmental conditions may also effect the outcome of a gram stain because these ultimately reflect on the chemical nature of the cell.

1. Prepare a heat fixed smear of the culture you wish to examine
2. Flood the smear with crystal violet (30 sec. to 2 min)
3. Quickly and gently wash off excess stain (2 seconds)
4. Flood the smear with Grams iodine (1 minute)
5. Decolorize with alcohol (10-20 seconds or until the excess alcohol which flow off the slide is colorless)
6. Quickly and gently wash off excess stain (2 seconds)
7. Flood the smear with safranin (30 sec to 2 min.)
8. Quickly and gently wash off excess stain (2 seconds)
9. Blot dry with bibulous paper
10. Examine your slide under the microscope. Record sketches of the organisms, size, color, morphology, and culture identification.

Gram stain and General Microscopy Troubleshooting

Problem	Probable Cause	Corrective Action
Slide is almost focused, but is hazy or unclear	Inadequate or no immersion oil	There should be enough oil to make a seal between the slide and the 100X objective
	Dirty Objective	Contact instructor to clean the objective
Slide is clear under 10X but cannot be focused under oil	Slide is upside down	Check slide and flip over if necessary
Organisms appear to have a halo around them or cannot be focused	Oil was added before slide was dry	Make a new slide. Allow slide to dry longer or blot more carefully before adding oil
Slide is in focus, then a shadow appears	Air bubble trapped by lens	Rotate objective back and forth
Floating material is seen	Objective scraped the smear	Clean objective; gently remove oil from slide and refocus more carefully
Nothing is visible on the slide	Smear too thin	Remake with a thicker smear
	Slide wiped to dry it	Blot slides; never wipe
	Smear was not heat fixed	Repeat; heat fix the smear
Excessive deposits of stain crystals on slide	Inadequate rinsing between steps	Repeat; wash off one stain completely before adding the next
	Stain was allowed to dry on slide before rinsing	Repeat; watch timing of staining steps more carefully
Gram positive organisms have odd angular shapes	Viewing crystal violet crystals	Repeat: Do not allow dye to dry on the slide. Do not allow crystal violet to remain on slide longer than suggested times
Gram negative organisms look more like needles than rods	Viewing safranin crystals
Specimen appears mixed with large oval cocci when it should be one organism	Yeast growing in the staining reagents	Repeat after the instructor has filtered the reagents
Gram positive cells appear gram negative	Overdecolorization	Decrease decolorizartion time
	Non-viable organisms	Use a fresher culture
	Failure to include mordanting step (iodine)	Be careful to follow each step in the proper order
Gram negative organism appears gram positive	Underdecolorization	Increase decolorization time
	Forgot decolorization step	Be careful to follow each step in the proper order
	Stain dried on smear
Mixture of gram positive and gram negative organisms from a pure culture	Smear is too thick	Remake smear
	Culture is contaminated	Follow proper aseptic technique

Assignment: Upon completion of the laboratory you should be able to:

1. State why the gram stain is said to be a differential stain
2. Describe the differences between a gram-positive and a gram-negative cell wall
3. State the mechanism of the Gram stain and why differential staining of bacterial cells occurs.
4. Have accurate labeled sketches of all of the specimens you examined. Included with the sketches should be a brief description of the specimen, the magnification under which it was viewed, and an approximation of the size.
5. Describe the conditions which may result in a gram-positive cell staining gram negative.
6. State the procedure for the gram stain
7. Perform a gram stain given all of the necessary materials and reagents
8. Determine if a bacterium is gram-positive or gram-negative when microscopically viewing the gram stain preparation and state the shape and arrangement of the organism.

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Forward to Lab 4: Pure Culture and Isolation of Organisms