Purpose:

To learn what is differential staining and its purposes
To perform three types of differential staining: Gram staining, Endospore staining and capsule staining
To understand their value when used to stain clinical specimen
To understand the different characteristics of bacteria and thus the differentiation in staining
To understand the basic characteristic of Gram (+) and Gram(-) bacteria and distinguish between them
To be familiar with the importance of endospores and capsule in bacteria
To have a background of the mechanisms of the dyes used in microbiological studies.
To learn dyes used at different staining methods in differential staining

Procedure:

A) Gram Staining
In this procedure different types of bacteria are used throughout the laboratory. These are:

Staphylococcus aureus,
Enterobacter aerogenes,
Bacillus substilis,
Salmonella anatum,
Escherichia coli,
Proteus vulgaris.

Our group (#7) used two of them: Escherichia coli and Proteus vulgaris. These were overnight cultures.
Remove a loopful of NB which contain E.coli and P.vulgaris and placed it on different slides separately and allow it to air dry.
Dry smear, and cover the bacteria on the slid with crystal violet and wait for 1 min.
Pour crystal violet and rinse gently with distilled water and then cover the smear with iodine for 1 min. Then pour off the dye and rinse gently with distilled water.
Allow 95% ethanol to flow over the smear using dropper. The crystal violet will wash off the smear, so we stopped washing till the smear is colorless. (It is important not to over decolorize otherwise Gram (+)’s will lose the violet stain. Then we immediately rinsed with distilled water)
Cover the smear with safranin and wait for 1 min and then wash with water and blot carefully with tissue paper.
Finally after the slide is air-dried, then observe the bacterial cells under 40X and 100X, pay attention to the cell size, shape and color.

B) Endospore staining
In this part of the experiment we stained the endospores of Bacillus substilis with Malachite green, (an old culture, kept for 3 days at 370C)

Prepare a smear of B. Subtilis, and since the dye used is carcinogenic deal the slides with gloves
Put the slide to tripod places in staining rack, and cover the smear with Malachite green and wait for 1 min
Passed the flame under the slide for many times still vapor was observed but not boiling
Cooled the slide and washed it with water, and then we apply safranin and wait for 1 min. After 1 min has passed rinse the slide with water and performe blotting
Finally observe slides under 40X and 100X

C) Capsule staining: use Klebsiella pneumoniae species to see capsule formation but be careful about pathogenecity of bacteria.

Put a drop of Indian ink to one edge of the slide, and after we obtain aseptically a loop of Klebsiella pneumonia culture and mixed with the ink.
Spread the ink with a second slide and apply it for air dry. And finally cover it with safranin for 1 min.
Finally rinse the dye with water and blot the slide and observe it under 100X.

Results:

Species
Observed colors
Expected colors
S.aureus
purple
purple
E.coli
pink/pink
pink
S.anatum
purple
pink
P.vulgaris
purple
pink
B.subtilis
pink/pink/pink
purple
E.aerogenes
pink/pink
pink

Species
Gram Staining(Expected)
S.aureus
positive(+)
E.coli
negative(-)
S.anatum
negative(-)
P.vulgaris
negative(-)
B.subtilis
positive(+)
E.aerogenes
negative(-)

Our group observed two bacteria for gram staining. First of Escherichia coli was rod shaped, pink colored. Due to pink color of it we conclude that it is Gram (-),d does not have any cell wall but have outer membrane. However, Proteus vulgaris was coccus and color after staining was purple so that we understand that it is Gram (+), it has cell wall. Expected result is also same with experimental results of our group but it is not true for B.subtilis observations. They saw pink color this may be due to high staining of Crystal violet and cause of degrade cell wall properties, and also reason for wrong observation might be that they used clear background and high amount of light so they thought that it is pink rather than purple. It is also possible some genetic problems or degradation or loss of peptidoglaycan at their cell wall so they can not show Gram (+).

For the endospore staining we used Bacillus subtilis and the result was failure because Bacillus subtilis is very small to see even at 100X oil immersion. However we know that cell having blackish dots one side of the cells should be endospore part of endospore formed bacteria and also vegetative part should be stain pink due to safranin.

For the capsule staining we observed shining part of capsule with careful observation but it is not certain that we saw correct thing because we did not use India ink and so that we could not stain surface with black effectively.

Discussion:

In this experiment, we have learned several basic molecular biology techniques and use of equipments such as differential staining techniques such as Gram staining, endospore staining and capsule staining.

Stains are chemical substances that make bacterial cells more visible by increasing the contrast between the cell and background. The dyes are usually organic molecules of complex. All dyes selectively absorb light of certain wavelengths and thus have a color. Many dyes useful for staining bacterial cells also specifically bind to the surface of bacterial cells. This category of dye is referred as positive stains. Some dyes do not bind to the surface of bacterial cells. These dyes called negative stains, they stain environment like Indian ink staining. With negative stains bacterial cells appear white. We use staining as help to provide information about cell morphology- size, shape and arrangement. In the case of Gram staining, it can also provide more detailed information about such cell propertied as the presence of a cell wall.

Gram staining is one of the examples of positive staining. Crystal violet (a positively charged) evidently binds to a negatively charged molecule, probably in the cell envelope or nucleic acid in the center of cell. Iodine must be added to form insoluble iodine-CV complexes. EtOH is used as a mordant, the Gram (+) bacteria with their thick peptidoglycan layers and with relatively little lipid decolorize slowly whereas the Gram (-) bacteria decolorize rapidly because of the ethanol dissolves their outer membrane lipid allowing the crystal violet-iodine complex to be released or because their thin peptidoglycan layer can not trap the complex. Finally, counterstaining with the safranin is done to make the Gram (-) cells visible in the microscope.

For our experiment we used S.aureus, E.coli, S.anatum, P.vulgaris, B.subtilis, E.aerogenes cultures. Most of the results are expected but there are also some unexpected results which most probably caused by some errors during staining and observation. Becasue purple color and pink color are very similar; we might see wrong color under microscope.

Another stain that has been used for years is the indirect or negative stain. The advantage of this stain is that it is the simplest and often quickest means of discovering cell shape and possibly refractive inclusions and endospores. It also does not distort bacteria, which may happen with Gram stains, since cells sometimes, shrink as a result of heat fixation. We used Schaffer-Fulton Technique for endospore staining of Bacillus substilis culture. Malachite Green is used as a primary stain then we apply steam to enhance penetration of the dye. Then we cooled and decolorize the slides by rinsing with water. Then we apply safranin as a counterstain to stain the colorless vegetative parts. At the end of the process spores appear in green and vegetative parts appear in red. Actually, we saw endospore parts black in general and vegetative part as red.

Principles of negative staining are as followings in case of capsule staining technique. We used a polysaccharide-containing material, in other words glycocalyx, lying outside of the bacterial cell to stain. It is related with pathogenecity because it aids attachments, contributes to immune system and it prevents dessication by binding some water. Capsules are largely water soluble and nonionic so they don’t bind to ordinary stains. Also heat activation can not used because it damages the capsule. So we used Indian ink for the background staining and safranin for staining the cell. This is actually not Indian ink but we could not find any Indian ink so that we tried to use usual, custom ink to stick cells to surface and stain surface black. As a result, we could not observe the capsule. Most probably the dye we used did not work effectively as effective as Indian ink.

In conclusion, these exercises were successful and they were a good and essential part of study and learn microbiology. New procedures were practiced, and further understanding of differential stainings, charachters of endospore, capsule, gram staining and principles of stainigs were gained.

REFERENCES:

TAXONOMY, Classification, Nomenclature, Laboratory Identification. Retrieved November 1, 2005 fromhttp://medic.med.uth.tmc.edu/path/00001458.htm
Staphylococcus. Retrieved November 1, 2005 from http://textbookofbacteriology.net/staph.html
Bacillus subtilis. . Retrieved November 1, 2005 from http://en.wikipedia.org/wiki/Bacillus_subtilis.