1 Experiment 6: Gram staining

Experiment 6: Gram Staining & Observation of Microorganisms

1. Abstract

This experiment is conducted to know how Gram staining is carried out. Gram staining is an empirical method of differentiating bacterial species into two large groups which are Gram-positive and Gram-negative bacteria based on the chemical and physical properties of their cell walls. The Gram stain is almost always the first step in the identification of a bacterial organism. In this experiment, Xanthomonas campestris is used as the specimen. Gram staining method is used to classify which group Xanthomonas campestris is belongs to and also to observe its morphology under the microscope. In this method, four types of dyes were being used which are crystal violet, iodine solution, decolourizer and safranin solution. All these four dyes have their own significant in Gram staining procedure. Unfortunately, after performing Gram staining onto the Xanthomonas campestris there was no bacterial image can be seen under the microscope. This situation might due to the Xanthomonas campestris have been washed away during the staining and rinsing process. However, Xanthomonas campestris is a Gram negative bacteria and red or pink stain should be observed under the microscope.

2. Introduction

In the second half of nineteenth century, scientists proved that specific bacterial organisms caused specific diseases, and the field of microbiology was on its way to becoming a distinct science. The microscope was also further developed during that time, and scientists were concerned with identifying and classifying bacteria. Most bacteria are difficult to see with a compound microscope, but can be seen when there is obvious contrast between the bacteria cells and their surrounding medium. Various dyes are used to stain cells so that they are more easily seen. As early as the late eighteenth century, scientists had developed some basic methods of staining cells to aid in their study and used natural substances such as saffron, which stained some parts of a cell. The discovery of synthetic dyes in the mid-1800s enabled scientists to utilize more colours to stain cells.

It is important for biologist to study the morphology and degree of motility of bacteria in order to identify and classify them in which this information can be obtained by observation of bacteria under the microscope. Gram staining was first introduced and named after the Danish bacteriologist who originally devised it in 1844, Hans Christian Gram. It is one of the most important staining techniques in microbiology. Besides Gram's stain, there is a wide range of staining methods available. The procedures for these other methods follow quite closely those of Gram's stain.

There are several stains being used in this method. The crystal violet stain is the primary stain, which stains everything in the smear blue. The Gram's iodine acts as a mordant that causes the crystal violet to penetrate and adhere to the gram-positive organisms. The acetone-alcohol mixture acts as the decolourizer that washes the stain away from everything in the smear except the gram-positive organisms. The safranine is the counter-stain that stains everything in the smear that has been decolorized.

1

2 Experiment 6: Gram staining

By using appropriate dyes, different parts of the cell structure such as capsules, flagella, granules, or spores can be stained. Staining techniques are widely used to visualize those components that are otherwise too difficult to see under an ordinary light microscope either because of the lack of colour contrast between the object under examination and the background or because of the limited resolving power of the light microscope. In addition, staining techniques are useful in detecting the presence or absence of certain cell components, thus allowing a relatively simple scheme of differentiation or identification of microorganisms. In this respect, Gram's stain ranks among the most important diagnostic tools in biological science.

3. Objectives

i. To develop the necessary skills in making slides.ii. To apply an appropriate methods of Gram staining.

iii. To know the importance of Gram staining methods in microbiology field.iv. To introduce morphology of bacteria.

4. Theory

In Gram staining (Gregersen, T., 1978) a species of bacteria can be classified as Gram positive and also Gram negative. The primary stain of the Gram's method is crystal violet. Crystal violet is sometimes substituted with methylene blue, which is equally effective. Gram positive bacteria retain crystal violet and hence appear deep violet in colour and gram negative bacteria lose the crystal violet and retain safranine and hence appear red in colour. The cell walls for gram-negative microorganisms have higher lipid content than gram-positive cells. Originally, both kinds of cells are penetrated by the crystal violet. Next, iodine solution is subsequently added as a mordant to form the crystal violet-iodine complex so that the dye cannot be removed too easily. This complex is extracted from the gram negative bacteria but not from gram positive bacteria by decolorizing agent which is alcohol.

The cell wall of gram positive bacteria consists of thick peptidoglycan layer and have no lipid outer membrane. These peptidoglycan layer become dehydrated by alcohol causing the pores in the walls to close and preventing the insoluble crystal violet-iodine complex from escaping. By contrast, gram negative bacteria has thin peptidoglycan layer with an outer lipid membrane. Alcohol readily penetrates this lipid rich outer membrane and extracts the crystal violet-iodine complex from the cell and these bacteria remain colourless, thus retaining the colour of safranine. This step is commonly referred to as fixing the dye. However, the subsequent treatment with the decolourizer, 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.

2

3 Experiment 6: Gram staining

As a result, the diffusion of the stain-iodine complex out from the cell is obstructed, and the cells remain stained. The actual mechanism of decolourization is currently not well understood and remains controversial. At any rate, if the decolourizer treatment is properly timed, there exists a period during which the crystal violet-iodine complex is effectively removed from the gram-negative cells but still retained in the gram-positive ones. Thus, the length of the decolourizer treatment is critical in clearly differentiating the gram-positive cells from the gram-negative cells. A prolonged exposure to the decolorizing agent will remove all the stain from both types of cells. The student is cautioned that some gram-positive cells lose the stain easily and therefore may appear gram negative.

5. Materials

i. Crystal violet ii. Iodine

iii. 95% ethanol iv. 0.25% safraninv. Oil

vi. Distilled watervii. Slide

viii. Microscope

6. Procedures

i. The smears are prepared and heated.ii. The smears are prepared on the slide and heated.

iii. The crystal violet is flooded for one minute.iv. The excess dye is poured off and washed gently in tap water and the slide is drained

against a paper towel.v. The smears are exposed to Gram’s iodine for one minute by washing with iodine, then

more iodine is added and left it on the smear for one minute.vi. The slide is washed with tap water and being drained carefully.

vii. The slide is washed with 95% alcohol for 30 seconds.viii. The slide is washed with tap water at the end of the 30 seconds to stop the

decolourization. Then it is being drained again.ix. The slide is counterstained with 0.25% safranin for 30 seconds.x. The slide then being washed, drained, blotted and examined under oil.

xi. The cells are sketched to show the morphology, grouping and relative sizes.

3

4 Experiment 6: Gram staining

7. Results

4

5 Experiment 6: Gram staining

8. Discussions

The Gram stain is an important tool for distinguishing between two main types of bacteria based on the differences of the structures of cell wall which are Gram-positive and Gram-negative. The bacterium used in this experiment is Xanthomonas campestris. The Gram staining procedures are used in this experiment to identify the group of bacteria Xanthomonas campestris belongs to and to observe its morphology under the microscope.

In this experiment, the Xanthomonas campestris on a microscope slide are heat-fixed and stained with a primary stain Crystal violet in which stains all bacterial cells blue. After that, Iodide solution is then added that allows the iodine to enter the cells and form a water-insoluble complex with the crystal violet dye. Iodine solution acts as a mordant in which it is able to dissolve the Crystal violet. The crystal violet and iodine bind to create a large molecule that is too big to exit the multiple layers of peptidoglycan in the Gram positive cells, so the purple colour becomes trapped.

The preparation is then treated with a decolourise solvent, in which the iodine-crystal violet complex is soluble. A decolourizer is applied in which it removes the purple stain from Gram-negative cells, leaving them clear. After rinsing the decolourizer, a secondary stain, safranin is then applied to impart a pink colour to the Gram-negative cells. After a final rinse, the slide can be viewed under oil immersion with a microscope.

The cells that retain the primary stain will appear blue or purple and are known as Gram positive. Cells that stain with the counterstain which is safranin will appear pink or red and are known as Gram negative. However, after the Xanthomonas campestris is stained and being observed under the microscope, there is no bacterial image can be seen. This situation might due to the bacteria, Xanthomonas campestris has been washed away during the rinsing of the dyes. So more precaution need to be taken during the staining on the slide and a proper method of staining is needed to avoid this mistakes.

Xanthomonas campestris is a Gram negative bacterium and through this experiment, the bacterium should have red or pink stain when it is being observed under the microscope. Gram negative bacteria (Salton, M. R. J, 1964) have a complex cell wall compare to Gram positive bacteria. The cell walls of Gram negative bacteria also have a thinner peptidoglycan layer with an outer lipid membrane. The absence of techoic acid in Gram negative also makes it different with Gram positive bacteria.

5

6 Experiment 6: Gram staining

Figure 8.1: The differences between Gram positive and Gram negative cell wall.

Although the Gram staining is the vital and almost the first steps towards identification of bacteria, this Gram staining also has some limitations. For example, some Gram-positive bacteria may lose the stain easily and therefore appear as a mixture of Gram-positive and Gram-negative bacteria (Gram-variable). When over-decolorized, even Gram positive bacteria may appear pink and when under-decolorized gram negative bacteria may appear Gram positive. The Gram reaction also depends on the age of the cell. Old cultures of Gram positive bacteria (where cell walls may be weakened) may readily get decolorized. Gram positive cells affected by cell wall active agents such as lysozyme or antibiotics may become Gram negative. Gram-positive bacteria such Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of these cells. In cultures of Bacillus, and Clostridium a decrease in peptidoglycan thickness during cell growth may cause some of them to appear Gram negative.

Certain group of bacteria can display variable response to the stain, which can be due to growth stress (e.g., unsuitable nutrients, temperatures, pHs, or electrolytes) that results in a number of nonviable, gram-negative cells in a gram positive culture, but certain bacterial species are known for their gram variability even under optimal growth conditions. Some bacteria tend to appear Gram negative when grown in acidic medium.

Loss of cell walls in Gram positive bacteria may render them Gram negative (L-forms). Bacteria totally devoid of cell wall (Mycoplasma) are always Gram negative. Bacteria such as Mycobacterium that have extra waxy content in their cell wall are difficult to stain. Small and slender bacteria such as Treponema, Chlamydia, Rickettsia are often difficult to stain by Gram's method. Gram positive bacteria that have been phagocytosed by polymorphs may also appear Gram negative.

In microbiology field, Gram staining has many benefits. It actually helps the microbiologist to differentiate between Gram-positive and Gram-negative organisms. Differentiation of bacteria into Gram positive and Gram negative is the first step towards classification and identification of bacteria. When new microorganisms are being found, it is important to study

6

7 Experiment 6: Gram staining

about its morphology and its cellular arrangement in order to classify them into the existence group. Thus, the Gram staining procedure is required to fulfil these needs. Not only have that, this method also helped to enhance the contrast when the microorganism are being observed under the microscope.

. 9. Conclusions

Throughout this experiment, a proper technique must be applied when performing this procedure so that the specimen on the slide is not being washed away during the rinsing of the dyes. This method has a lot of application in many field especially in microbiology, pharmaceutical and medicine field.

In pharmaceutical field for some instances, the chemical that make-up the cell wall also determines the penetrability of the wall by various drugs. Knowing if a bacterium is Gram positive or negative determines what type of antibiotic is suitable for treatment, as some antibiotics act against Gram positive bacteria (i.e., penicillin), while others act against Gram negative bacteria (i.e., tetracycline or streptomycin). Another important consideration is the fact that some Gram negative bacteria release endotoxins, which can be fatal. When pharmaceutical companies develop new antibacterial drugs, the Gram stain is the method by which scientists determine the effectiveness of the drug.

Besides that, this Gram stain method was also applied to the photometric characterization (Durtschi and J. D,2005) of aquatic bacterial populations with a charge-coupled device camera and an image analyzer. Escherichia coli and Bacillus subtilis were used as standards of typical gram-negative and gram-positive bacteria, respectively. A mounting agent to obtain clear images of Gram-stained bacteria on Nuclepore membrane filters was developed. The bacterial stainability by the Gram stain was indicated by the Gram stain index (GSI), which was applicable not only to the dichotomous classification of bacteria but also to the characterization of cell wall structure. The GSI spectra of natural bacterial populations in water with various levels of eutrophication showed a distinct profile, suggesting possible staining specificity that indicates the presence of a particular bacterial population in the aquatic environment.

Not only have that, the Gram stain is not just an infallible tool for diagnosis, identification, or phylogeny, however. It is of extremely limited use in environmental microbiology, and has been largely superseded by molecular techniques even in the medical microbiology lab. Some organisms are Gram-variable that means they may stain either negative or positive and some organisms are not susceptible to either stain used by the Gram technique. In a modern environmental or molecular microbiology lab, most identification is done using genetic sequences and other molecular techniques, which are far more specific and information-rich than differential staining.

In medicine field, Gram stains are performed on body fluid or biopsy when infection is suspected. It yields results much more quickly than culture, and is especially important when

7

8 Experiment 6: Gram staining

infection would make an important difference in the patient's treatment and prognosis; examples are cerebrospinal fluid for meningitis and synovial fluid for septic arthritis. Being able to identify bacteria by stain result/photo, or negative stain and history is a typical Medical Boards question. Special Stains and Growth Media, such as India Ink for Cryptococcus and actual appearance of haemolytic streps on growth plate are seen on test questions.

On medical field also observation of bacteria in clinical specimens using Gram staining method provides a vitals clue in the diagnosis of infectious diseases. Empirical choice of antibiotics also can be made on the basis of Gram stain’s report. Gram staining also useful in estimation total count of bacteria. Last but not least, choice of culture media for inoculation also is made empirically based on Gram’s stain report.

In conclusions, Gram staining method provides much application in many different fields such as microbiology and medicine field. a proper technique is also important in carrying out Gram staining method because this methods also has several limitations. It is also to ensure the accuracy on the image observed under the microscope.

10. Recommendations

i. The rinsing process of dyes needs to be carried out slowly to avoid the specimen being washed away.

ii. Watch out the timing (if it is needed) as over timing it will cause inaccuracy in the image observed.

iii. Make sure the inoculationg loop is being flamed before the bacteria are being transferred onto the slide.

iv. Do not over heat the slide before dying process take place. It would cause the bacteria died.

11. References

i. Salton, M. R. J., The Bacterial Cell Wall, Elsevier Press, Amsterdam, 1964.ii. Bartholomew, J. W. and Finkelstein, H., Relationship of cell wall staining to Gram

differentiation, J. Bacteriol., 75, 77, 1958.iii. Gregersen, T., Rapid method for distinction of gram-negative from gram-positive

bacteria, Eur. J. Appl. Microbiol. Biotechnol., 5, 123, 1978.iv. Durtschi, J. D, Erali, M., Bromley, L K., Herrmann, M. G, Petti, C. A, Smith, R. E,

Voelkerding, K. V (2005). Increased sensitivity of bacterial detection in cerebrospinal fluid by fluorescent staining on low-fluorescence membrane filters. J Med Microbiol 54: 843-850

8

9 Experiment 6: Gram staining

12. Appendices

9

Plate 11.1: The transfer of specimen onto the slide.

Plate 11.2: The slide is being heat-fixed.

Plate 11.3: The process of staining.

Plate 11.4: The slide is left to be dried.

10 Experiment 6: Gram staining

1. What is the purpose of staining bacteria?The purpose of staining bacteria is to identify and classify bacteria into their major groups which are Gram positive bacteria and Gram negative bacteria. Not only that, through this method we can enhance the contrast for viewing under the microscope and to reveal the morphology and different properties of microorganisms.

2. List and describe the three basic bacterial shapes.a) Cocci

Are roughly spherical Diplococcic arise when cocci divide and remain together to form pairs Tetrad-group of 4 Long chain of cocci (streptococcus) results when cells adheres after repeated

division in one plane. Staphylococcus- cluster

b) Rod Sometimes called bacillus. The shape of the rod’s end may be flat, rounded, cigar-shaped or bifurcated. Diplobacillus-attached in pair Streptobacillus-in form of a chain

c) Spiral Vibrios most closely resembles rods, as they are comma-shaped. Spiral-shaped can be either classified as spirilla.

3. What are the differences between a simple stain and a differential stain?

Simple stains use only one dye that stains the cell wall of bacteria much like dying eggs at Easter. Differential stains use two or more stains and categorize cells into groups. Both staining techniques allow the detection of cell morphology, or shape, but the differential stain provides additional information concerning the cell.

10

11 Experiment 6: Gram staining

4. What is the most common differential stain used in microbiology?

The most common differential stain used in microbiology is the Gram stain.

5. What is the basis for Gram stain results between different bacteria?

The colour of the stain which reflects the major group of the bacteria.

6. List the reagents used in the Gram stain and tell the function of each.a) Crystal violet

Acts as the primary stain. Can also be used as a simple stain because it colours the cell wall of any bacteria.

b) Iodine Acts as a mordant. This reagent reacts with the crystal violet to make a large crystal that is not easily

washed out of the cell. At this point in the staining process all cells will be the same colour.

c) Decolourizer( alcohol and acetone)

The difference in the cell wall structure is displayed by the use of the decolorizer. The decolorizer does not affect those cell walls composed primarily of

peptidoglycan but those with the lipid component will have large holes develop in the cell wall where the lipid is dissolved away by the acetone and alcohol.

These large holes will allow the crystal violet-iodine complex to be washed out of the cell leaving the cell colourless.

d) Safranin Applied to the cells which will dye the colourless cells.

7. What would be the proper way to report each of the following if they had been Gram stained?

a) Purple (blue), round cells Gram positive cocci and rod-shaped

b) Pink (red), rod-shaped cell Gram negative bacilli

c) Pink (red), round cells Gram positive cocci and rod-shaped

d) Purple (blue), rod-shaped cells Gram positive bacilli

11