How To Do Gram Staining
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Observation of microorganism under microscope can be improved by using certain processes and techniques such as the staining. Staining is an important step to observe microorganisms more clearly, to differentiate between microorganisms as well as to differentiate parts in microorganism (Bagyaraj et al, 2005). The identification, morphology, some extracellular and intracellular components of microorganisms can be determined and detected through the staining. Many microorganisms difficult to be observed under microscope due to their colourless appearance and semitransparent properties as their refractive index almost same as surroundings (Patil et al, 2008). The stain improves contrast for visualizing microorganisms. Staining process can be explained either as physical, chemical reaction or combination of the both reaction.
There are different types of staining such as the simple stain, differential stain and special stain. Simple stain can be used for observing certain basic structures as well as the shape of microorganisms. Differential stain while can be used in distinguishing between different types of microorganisms. Special stain on the other hand can be used for identifying specific structures in the microorganisms such as the flagella (Frey & Price, 2003). Gram-stain is one of the commonly used differential stains. The Gram-staining process discovered in 1882 (published 1884) by Hans Christian Gram, a Danish bacteriologist and plays an important role in the classifying the bacteria. Gram-staining is usually the first step in identification bacteria and can be used in characterizing bacteria. Bacteria species can be separated into two large groups, which are the Gram-positive and Gram-negative groups through the Gram-staining (Sridhar Rao, n.d.). This process also important in clinical laboratory such as to examine and identify bacteria responsible for certain diseases.
Staining process requires the preparation of smear that contains a thin layer of bacteria. The preparation of smear involves spreading and fixing of microorganisms on the microscope slide. Use of smear prevents microorganisms from being washing away with stain (Vasanthakumari, 2009). Besides the smear, there are four important components in the Gram stain process, which are the primary stain, mordant, decolourizing agent as well as the counterstain that used in sequences. The primary stains usually basic dye such as crystal violet that reacts with acidic component of cell and causes all the bacteria to be stained with the crystal violet or purple. The other dye like the methyl violet can also be used. The other component, mordant in the Gram stain refers to iodine. Mordant is chemical that increases affinity of the stain to the microorganisms and also their coating, making certain structures thicker for easier observation under microscope. The decolorizing agent decolorizes dye from cell that already being stained (Rajan, 2005). The degree of decolorization different in bacteria depends on their chemical components. Decolourization agent commonly refers to ethanol or other solution like acetone or mixture of acetone and ethyl alcohol. Counterstain while is another basic dye that important in giving new colour for cells that decolourized. Counterstain can be the safranin (used in this practical) or the carbon fuchsin.
The Gram stain (differential stains) gives different colour for different types of bacteria. The colour is the one that determine whether the bacterium is Gram positive or Gram negative. The Gram positive bacteria resist decolourization and give result of crystal violet or purple colour (primary stain). Gram-negative bacteria decolorize and give red or pink colour as it takes up counterstain (Ananthanarayan & Paniker, 2006). The difference in result is due to the differences in the cell wall structure or composition of bacteria that causes the different in the reaction with the series of reagents in Gram staining (Talaro, 2007).
Preparation of Staining Reagents:
Solution A: Crystal violet 2.0g
Ethanol, 95% (v/v) 20 ml
Solution B: Ammonium oxalate 0.8g
Distilled water 80 ml
Solution A and B mixed.
Iodine 1.0 g
Potassium iodide 2.0 g
Distilled water 300 ml
Iodine and potassium blended with mortar, distilled water added during blending until iodine dissolved.
Ethanol, 95% (v/v)
Safranin 0.25 g [2.5 %(w/v)]
Ethanol 10 ml [9.5% (v/v)]
Distilled water 90 ml
Escherichia coli in broth culture
Escherichia coli in agar culture
Bacillus sp. in broth culture
Bacillus sp. in agar culture
Staphylococcus aureus in broth culture
Actinomycetes sp. in broth culture
Actinomycetes sp. in agar culture
Preparation of smear:
For culture taken from liquid medium (broth), 1 drop of culture to be examined was transferred by using inoculation loop onto a slide and spread to from circular smear. For culture taken from solid medium (agar), one drop of distilled water first dispensed on the slide. The single colony then spread on the water to form circular smear.
The slide was heat-fixed with flame.
The slide was placed on the rack.
1-2 drops of crystal violet was dropped on the smear and left for 2 minutes.
The crystal violet was rinsed off with distilled water for 2 seconds.
Iodine solution was dropped and left for 2 minutes.
The iodine solution was rinsed off with distilled water for 2 seconds.
The smear was decolorized by washing with ethanol (95%v/v) for less than 10 seconds. The ethanol then rinsed off with distilled water for 10 seconds.
Safranin solution was dropped on the smear for 10 seconds.
The red-coloured safranin was rinsed-off with distilled water.
The side was dried using Kimwipe or air-dry.
The slide was observed under the microscope.
1(a) Broth culture (zoom in).
1(b) Agar plate (zoom in).
Figure 1: Microscopic image of Escherichia coli under total magnification of 400Ã- from different culture
(B) Bacillus species
2(a) Broth culture (zoom in).
2(b) Agar plate (zoom in).
Figure 2: Microscopic image of Bacillus sp. under total magnification of 400Ã- from different cultures.
(C) Staphylococcus Aureus
Figure 3: Microscopic image of Staphylococcus aureus under total magnification of 400Ã- from broth culture (zoom in).
(D) Actinomycetes species
C:\Users\michelle\Documents\UMS MICROBIOLOGY\PHOTOSS\1.JPG G:\DCIM\101NIKON\DSCN1760.JPG
4(a) Broth culture (zoom in) under total magnification of 400Ã-.
4(b) Agar plate (zoom in) under total magnification of 400Ã-.
Figure 3: Microscopic image of Actinomycetes sp. under different magnification from different culture.
Table 1: The result of Gram stain on different microorganism
Type of microorganisms
Shape of the microorganisms
Colour stained on microorganisms
Gram positive or Gram negative
Escherichia coli (broth culture)
Bacillus or Rod-shaped
Escherichia coli (agar plate)
Bacillus or Rod-shaped
Bacillus sp. (broth culture)
Bacillus or Rod-shaped
Bacillus sp. (agar plate)
Bacillus or Rod-shaped
Coccus or round-shaped
Actinomycetes sp. (broth culture)
Actinomycetes sp. (agar plate)
For every bacterium studied, a smear is first prepared as the smear enables Gram staining to be done without washing away bacteria together with stain. The spreading process (for both broth and agar culture) enables the distribution of bacteria on slides so that suitable density of bacteria can be found on the slide. This increases chance of individual bacteria to be observed under microscope (Port, 2009). The microorganisms from agar first suspended in distilled water before spreading. Without spreading, bacteria may be too concentrated, crowded and overlapped (in clumps), making the observation to be difficult. The slide was heat fixed after drying. Heating enables coagulation and precipitation of protein of bacteria to occurs, hence fix the bacteria on slide. The bacteria killed and adhere to the surface. Fixation makes the bacteria rigid, immobile, increased permeability and affinity to staining. This also prevents the autolysis process of bacteria (Aneja, 2003). During the fixation process, slides not be placed directly above the heat or passed through too many times as overheat may causes changes in the shape and hence cause the distortion of the microorganisms. At the same time, less heat supplied may cause the microorganisms do not fix firmly. Before heat fix, the slide is allowed to dry completely as wet bacterial suspension may create aerosol (Shimeld, 1999).The presence of water may also cause over heating.
The crystal violet added as the primary stain. Crystal violet is basic dye and has affinity for cell structures that are acidic such as the protoplasm. Crystal violet is added to stain everything on slide or to stain all bacteria (Gram positive or Gram negative). This is same for all the seven samples. Crystal violet dye enters the cells and stained with crystal violet colour. It was suggested that the aqueous dye dissociated into CV+ ion and chloride, Cl- ion (Hussey & Smith, n.d.). The positively charged ion binds to the negatively charged components in cell after penetrating the cell wall and cell membrane, hence giving the purple colour. The extra crystal violet dye that not binds to cell is cleared by distilled water. Addition of iodine in next step enables the crystal violet dye to further fix and adhere to organisms (Medical Education Division, 2006). This is due to the formation of complex between iodine and dye ion (CV-I complex) as the negatively charged iodine ion (I- or I3- ion) binds to the positively charged ion of dye (CV+ ion) in cytoplasm and hence bacteria appeared as violet colour (Vasanthakumari, 2009). The solubility of the dye decreased during the process as the ions bind to organisms. Iodine acts as mordant as it increases affinity of crystal violet stain to organisms.
The addition of 95% ethanol as decolourizer enables the lipid to be extracted or dissolved from the cell wall for the Gram negative bacteria like the Escherichia coli. Gram negative bacteria have an outer membrane that constitutes most of the cell wall, also known as lipopolysaccharide layer (LPS) in cell wall (Clark et al, 2009). This is a lipid bilayer structure that differs from cytoplasmic membrane. This layer not only made up of phospholipids and protein, but also polysaccharides that not commonly found in cytoplasmic membrane. Polysaccharide portion made up of core polysaccharides and O-polysaccharides while the lipid portion made up of lipid A which then bind to the core polysaccharides. This LPS layer is located outside a thin layer of peptidoglycan. The outer membrane gives rises to high lipid composition in the cell wall. Decolourizer dissolve off lipid, hence increases the permeability of cell wall which eventually enables the crystal violet-iodine complex to be lost together with the lipid.
The cell wall (murein layer) of Gram positive layer while has no outer membrane but have thick, cross-linked and multi-layered peptidoglycan. Teichoic acids, the phosphorylated polyalcohol can be found embedded in peptidoglycan layers. These acids can be found bonded to muramic acid residues in peptidoglycan. Lipoteichoic acid which refers to the teichoic acids that binds to the lipids of membrane can also be found in Gram positive bacterial cell wall. In certain actinobacteria, structure called mycolic acids also can be found. The lack of outer membrane gives rises to low lipid composition in cell wall. Hence, the action of decolorizer on Gram positive bacteria (Bacillus sp., Staphylococcus aureus and Actinomycetes sp.) causes dehydration of cell wall due to the thick peptidoglycan and the composition of lipid available to be dissolved is low. This eventually decreases cell wall permeability, closing pores on cell wall and hence retain the crystal violet-iodine complex inside (Differential staining: The Gram Stain, n.d.). As the cell shrinks, the complex trapped in the thick peptidoglycan and hence cells do not decolourized. After this process, E. coli is in colourless as the crystal-violet iodine complex loses while Bacillus sp., Staphylococcus aureus and Actinomycetes sp. still in purple colour.
Ethanol was not added for more than 30 seconds. Over decolourization can cause the stain of Gram positive bacteria to decolourize and appears as Gram negative (Betts et al, 2003). Under decolourization (too short) also avoided as it can cause dye to be removed incompletely from Gram negative bacteria. Both situations can give false results. After decolorization, smear was washed with distilled water for 15 second to completely stop the decolourization process. The counterstain, safranin solution then stained the E. coli that is colourless with the red colour. Safranin is basic dye (cationic ion) carry the positive dye ion, chromophore that attached to acidic cell structures (negatively charged) such as the protoplasm. Basic dye also attached to other negatively charged macromolecules like proteins and nucleic acid (Archunan, 2004). Both the Gram positive and Gram negative bacteria took up the counterstain but the colour of Gram Positive do not change much as it already stained with purple. For every dye, there is different period of time for staining. This is to prevent over or under stain that may results in inaccurate result.
From the observation, Escherichia coli stained red and give accurate result of Gram negative. The shape of E. coli can be observed as rod shape. Bacillus sp., Staphylococcus aureus and Actinomycetes sp. while shows results of Gram positive as all are stained with purple colour. The shapes observed are respectively rod-shaped, round-shaped and in mycelial. For Staphylococcus aureus, the cocci shape is sticked together in clumps or amorphous sheet and not separated. For E. coli, bacillus sp. and staphylococcus aureus, two samples are taken, one from the broth and one from the agar. Both the samples show the same results. The difference is on the amount of microorganisms observed. Bacillus sp., for example, that taken from agar plate is very crowded. This is because the each colony taken contains a number of microorganisms. It is more difficult to be observed the shape of the organisms. However, the colour stained can be observed clearly. For the broth culture, individual organisms and the shape as well as the colour can be observed more clearly.
Gram staining is important in differentiating Gram positive and Gram negative bacteria in which the Gram positive bacteria stained purple colour while Gram negative organisms stained pink. Escherichia coli is Gram negative while bacillus sp., staphylococcus aureus and actinomycetes are Gram positive bacteria.
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