ExerciseⅣ Gram Stain
Materials per Student
18 to 24 hour tryptic soy broth cultures of formalinized（1 ml of concentrated formalin per 10 ml of culture）Staphyloccusaureus，Escherichia coli，and a mixture of S.aureus and E.coli solutions of crystal violet，Gram's iodine（2 g potassium iodide in 300 ml distilled water plus 1 g iodine crystals），95%ethanol and/or isopropanol-acetone mixture（3∶1 v/v），and safranin clean glass slides；inoculating loop；bunsen burner；bibulous paper；microscope；lens paper and lens cleaner；immersion oil；staining rack
Learning Objectives
Each student should be able to：
1.Learn the proper procedure for preparing a bacterial smear.
2.Understand the biochemistry underlying the Gram stain.
3.Understand the theoretical basis for differential staining procedures.
4.Perform a satisfactory Gram stain.
5.Differentiate a mixture of bacteria into gram positive and gram-negative cells.
Principles
In making a smear，bacteria from either a broth culture or an agar slant or plate may be used.If a slant or plate is used，a small amount of bacterial growth is transferred to a drop of water on a glass slide[Figure 1（a）]and mixed.The mixture is then spread out evenly over a large area on the slide[Figure 1（b）].
One of the most common errors in smear preparation from agar cultures is the use of too large an inoculum.This invariably results in the occurrence of large aggregates of bacteria piled on top of one another.If the medium is liquid，place one or two loops of the medium directly on the slide[Figure 1（c）]and spread the bacteria over a large area[Figure 1（d）].Allow the slide to air dry at room temperature[Figure 1（e）].After the smear is dry，the next step is to attach the bacteria to the slide by heat-fixing.This is accomplished by gentle heating[（Figure 1（f）]，passing the slide several times through the hot portion of the flame of a Bunsen burner.Most bacteria can be fixed to the slide and killed in this way without serious distortion of cell structure.
Simple staining depends on the fact that bacteria differ chemically from their surroundings and thus can be stained to contrast with their environment.Bacteria also differ from one another chemically and physically and may react differently to a given staining procedure.This is the principle of differential staining.Differential staining can distinguish between types of bacteria.The Gram stain（named after Christian Gram，Danish scientist and physician，1853- 1938）is the most useful and widely employed differential stain in bacteriology.It divides bacteria into two groups—gram negative and gram positive.
Figure 1 Bacterial Smear Preparation
The first step in the procedure involves staining with the basic dye crystal violet.This is the primary stain.It is followed by treatment with an iodine solution，which functions as a mordant；that is，it increases the interaction between the bacterial cell and the dye so that the dye is more tightly bound or the cell is more strongly stained.The smear is then decolorized by washing with an agent such as 95% ethanol or isopropanol-acetone.Gram-positive bacteria retain the crystal violet-iodine complex when washed with the decolorizer，whereas gramnegative bacteria lose their crystal violet-iodine complex and become colorless.Finally，the smear is counterstained with a basic dye，different in color than crystal violet.This counterstain is usually safranin.The safranin will stain the colorless，gram-negative bacteria pink but does not alter the dark purple color of the gram-positive bacteria.The end result is that gram-positive bacteria are deep purple in color and gram-negative bacteria are pinkish to red in color（Figure 2）.Corresponding book color chart NO.3 to 5.
Light micrograph（×100）of a Gram stained mixture of gram-positive Staphylococcus aureus（purple cocci）and gram-negative Escherichia coli（pink rods）
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Figure 2 Gram Stain
The Gram stain does not always yield clear results.Species will differ from one another in regard to the ease with which the crystal violet-iodine complex is removed by ethanol.Gram-positive cultures may often turn gram negative if they get too old.Thus，it is always best to Gram stain young，vigorous cultures rather than older ones.Furthermore，some bacterial species are gram variable.That is，some cells in the same cuture will be gram positive and some，gram negative.Therefore，one should always be certain to run Gram stains on several cultures under carefully controlled conditions in order to make certain that a given bacterial“strain”is truly gram positive or gram negative.
Indistinct Gram-stain results can be confirmed by a simple test using KOH.Place a drop of 10% KOH on a clean glass slide and mix with a loopful of bacterial paste.Wait 30 seconds，then pull the loop slowly through the suspension and up and away from the slide.A gram-negative organism will produce a mucoid string；a gram-positive organism remains fluid.
Procedure for Traditional Gram-Stain Technique
1.Prepare heat-fixed smears of E.coli，S.aureus，and the mixture of E.coli and S.aureus.
2.Place the slides on the staining rack.
3.Flood the smears with crystal violet and let stand for 30 seconds[Figure 3（a）
].
4.Rinse with water for 5 seconds[Figure3（b）].
5.Cover with Gram's iodine mordant and let stand for 1 minute[Figure 3（c）].
6.Rinse with water for 5 seconds[Figure 3（d）].
7.Decolorize with 95% ethanol for 15 to 30 seconds.Do not decolorize too long.Add the decolorizer drop by drop until the crystal violet fails to wash from the slide[Figure 3（e）].Alternatively，the smears may be decolorized for 30 to 60 seconds with a mixture of isopropanol-acetone（3∶1 v/v）.
8.Rinse with water for 5 seconds[Figure 3（f）].
9.Counterstain with safranin for about 60 to 80 seconds[Figure 3（g）].Safranin preparations vary considerably in strength，and different staining times may be required for each batch of stain.
10.Rinse with water for 5 seconds[Figure 3（h）].
11.Blot dry with bibulous paper[Figure 3（i）]and examine under oil immersion.Gram-positive organisms stain blue to purple；gram-negative organisms stain pink to red.There is no need to place a coverslip on the stained smear.See Figure 3 for an example of gram-positive and gram negative bacteria.
Figure 3 Gram-stain Procedure