How To Do Gram Staining Biology

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Observation of micro-organism under microscope can be improved by utilizing certain procedures and techniques such as the staining. Staining is an of import measure to detect micro-organisms more clearly, to distinguish between micro-organisms every bit good as to distinguish parts in micro-organism ( Bagyaraj et al, 2005 ) . The designation, morphology, some extracellular and intracellular constituents of micro-organisms can be determined and detected through the staining. Many micro-organisms hard to be observed under microscope due to their colourless visual aspect and semitransparent belongingss as their refractile index about same as milieus ( Patil et al, 2008 ) . The discoloration improves contrast for visualising micro-organisms. Staining procedure can be explained either as physical, chemical reaction or combination of the both reaction.

There are different types of staining such as the simple discoloration, differential discoloration and particular discoloration. Simple discoloration can be used for detecting certain basic constructions every bit good as the form of micro-organisms. Differential discoloration while can be used in separating between different types of micro-organism. Particular discoloration on the other manus can be used for placing specific constructions in the micro-organisms such as the scourge ( Frey & A ; Price, 2003 ) . Gram-stain is one of the commonly used differential discolorations. The Gram-staining procedure discovered in 1882 ( published 1884 ) by Hans Christian Gram, a Danish bacteriologist and plays an of import function in the sorting the bacterium. Gram-staining is normally the first measure in designation bacteriums and can be used in qualifying bacteriums. Bacteria species can be separated into two big groups, which are the Gram-positive and Gram-negative groups through the Gram-staining ( Sridhar Rao, n.d. ) . This procedure besides of import in clinical research lab such as to analyze and place bacteriums responsible for certain diseases.

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Staining procedure requires the readying of vilification that contains a thin bed of bacteriums. The readying of smear involves distributing and repairing of micro-organisms on the microscope slide. Use of smear prevents microorganisms from being rinsing off with discoloration ( Vasanthakumari, 2009 ) . Besides the vilification, there are four of import constituents in the Gram discoloration procedure, which are the primary discoloration, mordant, bleaching agent every bit good as the counterstain that used in sequences. The primary discolorations normally basic dye such as crystal violet that reacts with acidic constituent of cell and causes all the bacteriums to be stained with the crystal violet or purple. The other dye like the methyl violet can besides be used. The other constituent, mordant in the Gram discoloration refers to iodine. Mordant is chemical that increases affinity of the discoloration to the micro-organisms and besides their coating, doing certain constructions thicker for easier observation under microscope. The bleaching agent decolorizes dye from cell that already being stained ( Rajan, 2005 ) . The grade of decolorization different in bacteriums depends on their chemical constituents. Decolourization agent normally refers to ethanol or other solution like propanone or mixture of propanone and ethyl intoxicant. Counterstain while is another basic dye that of import in giving new coloring material for cells that decolourized. Counterstain can be the saffranine ( used in this practical ) or the C fuchsin.

The Gram discoloration ( differential discolorations ) gives different coloring material for different types of bacterium. The coloring material is the 1 that determine whether the bacteria is Gram positive or Gram negative. The Gram positive bacteriums resist decolourization and give consequence of crystal violet or purple coloring material ( primary discoloration ) . Gram-negative bacteriums bleach and give ruddy or tap coloring material as it takes up counterstain ( Ananthanarayan & A ; Paniker, 2006 ) . The difference in consequence is due to the differences in the cell wall construction or composing of bacteriums that causes the different in the reaction with the series of reagents in Gram staining ( Talaro, 2007 ) .

Preparation of Staining Reagents:

Crystal violet

Solution A: Crystal violet 2.0g

Ethanol, 95 % ( v/v ) 20 milliliter

Solution B: Ammonium oxalate 0.8g

Distilled H2O 80 milliliter

Solution A and B mixed.


Iodine 1.0 g

Potassium iodide 2.0 g

Distilled H2O 300 milliliter

Iodine and K blended with howitzer, distilled H2O added during intermixing until iodine dissolved.

Decolorization dissolver

Ethanol, 95 % ( v/v )


Safranin 0.25 g [ 2.5 % ( w/v ) ]

Ethanol 10 milliliters [ 9.5 % ( v/v ) ]

Distilled H2O 90 milliliter


Glass slide

Escherichia coli in broth civilization

Escherichia coli in agar civilization

Bacillus sp. in broth civilization

Bacillus sp. in agar civilization

Staphylococcus aureus in broth civilization

Actinomycetes sp. in broth civilization

Actinomycetes sp. in agar civilization


Bunsen burner


Distilled H2O

Inoculation cringle


Preparation of vilification:

For civilization taken from liquid medium ( broth ) , 1 bead of civilization to be examined was transferred by utilizing vaccination cringle onto a slide and spread to from round vilification. For civilization taken from solid medium ( agar ) , one bead of distilled H2O foremost dispensed on the slide. The individual settlement so spread on the H2O to organize round vilification.

The slide was heat-fixed with fire.


The slide was placed on the rack.

1-2 beads of crystal violet was dropped on the vilification and left for 2 proceedingss.

The crystal violet was rinsed off with distilled H2O for 2 seconds.

Iodine solution was dropped and left for 2 proceedingss.

The iodine solution was rinsed off with distilled H2O for 2 seconds.

The vilification was decolorized by rinsing with ethyl alcohol ( 95 % v/v ) for less than 10 seconds. The ethyl alcohol so rinsed off with distilled H2O for 10 seconds.

Safranin solution was dropped on the vilification for 10 seconds.

The red-coloured saffranine was rinsed-off with distilled H2O.

The side was dried utilizing Kimwipe or air-dry.

The slide was observed under the microscope.


( A ) Escherichia coli


1 ( a ) Broth civilization ( rapid climb in ) .

1 ( B ) Agar home base ( rapid climb in ) .

Figure 1: Microscopic image of Escherichia coli under entire magnification of 400A- from different civilization

( B ) Bacillus species


2 ( a ) Broth civilization ( rapid climb in ) .

2 ( B ) Agar home base ( rapid climb in ) .

Figure 2: Microscopic image of Bacillus sp. under entire magnification of 400A- from different civilizations.

( C ) Staphylococcus Aureus


Figure 3: Microscopic image of Staphylococcus aureus under entire magnification of 400A- from broth civilization ( rapid climb in ) .

( D ) Actinomycetes species

Degree centigrades: UsersmichelleDocumentsUMS MICROBIOLOGYPHOTOSS1.JPG G: DCIM101NIKONDSCN1760.JPG

4 ( a ) Broth civilization ( rapid climb in ) under entire magnification of 400A- .

4 ( B ) Agar home base ( rapid climb in ) under entire magnification of 400A- .

Figure 3: Microscopic image of Actinomycetes sp. under different magnification from different civilization.

Table 1: The consequence of Gram stain on different micro-organism

Type of micro-organisms

Shape of the micro-organisms

Colour stained on micro-organisms

Gram positive or Gram negative

Escherichia coli ( broth civilization )

Bacillus or Bacillar


Gram negative

Escherichia coli ( agar home base )

Bacillus or Bacillar


Gram negative

Bacillus sp. ( broth civilization )

Bacillus or Bacillar


Gram positive

Bacillus sp. ( agar home base )

Bacillus or Bacillar


Gram positive

Staphylococcus aureus

Coccus or round-shaped


Gram positive

Actinomycetes sp. ( broth civilization )



Gram positive

Actinomycetes sp. ( agar home base )



Gram positive


For every bacteria studied, a vilification is foremost prepared as the smear enables Gram staining to be done without rinsing off bacteria together with discoloration. The distributing procedure ( for both broth and agar civilization ) enables the distribution of bacteriums on slides so that suited denseness of bacteriums can be found on the slide. This increases opportunity of single bacteriums to be observed under microscope ( Port, 2009 ) . The micro-organism from agar foremost suspended in distilled H2O before distributing. Without distributing, bacteriums may be excessively concentrated, crowded and overlapped ( in bunch ) , doing the observation to be hard. The slide was heat fixed after drying. Heating enables curdling and precipitation of protein of bacteriums to happen, therefore fix the bacteriums on slide. The bacterium killed and adhere to the surface. Fixation makes the bacteriums stiff, immobile, increased permeableness and affinity to staining. This besides prevents the self-digestion procedure of bacteriums ( Aneja, 2003 ) . During the arrested development procedure, slides non be placed straight above the heat or passed through excessively many times as overheat may do alterations in the form and hence cause the deformation of the micro-organism. At the same clip, less heat supplied may do the micro-organisms do non repair steadfastly. Before heat hole, the slide is allowed to dry wholly as wet bacterial suspension may make aerosol ( Shimeld, 1999 ) .The presence of H2O may besides do over warming.

The crystal violet added as the primary discoloration. Crystal violet is basic dye and has affinity for cell constructions that are acidic such as the living substance. Crystal violet is added to stain everything on slide or to stain all bacteriums ( Gram positive or Gram negative ) . This is same for all the seven samples. Crystal violet dye enters the cells and stained with crystal violet coloring material. It was suggested that the aqueous dye dissociated into CV+ ion and chloride, Cl- ion ( Hussey & A ; Smith, n.d. ) . The positively charged ion binds to the negatively charged constituents in cell after perforating the cell wall and cell membrane, therefore giving the violet coloring material. The excess crystal violet dye that non binds to cell is cleared by distilled H2O. Addition of I in following measure enables the crystal violet dye to farther hole and adhere to organisms ( Medical Education Division, 2006 ) . This is due to the formation of complex between I and dye ion ( CV-I composite ) as the negatively charged iodine ion ( I- or I3- ion ) binds to the positively charged ion of dye ( CV+ ion ) in cytol and hence bacteriums appeared as violet coloring material ( Vasanthakumari, 2009 ) . The solubility of the dye decreased during the procedure as the ions bind to organisms. Iodine acts every bit black as it increases affinity of crystal violet discoloration to organisms.

The add-on of 95 % ethanol as decolourizer enables the lipoid to be extracted or dissolved from the cell wall for the Gram negative bacteriums like the Escherichia coli. Gram negative bacteriums have an outer membrane that constitutes most of the cell wall, besides known as lipopolysaccharide bed ( LPS ) in cell wall ( Clark et Al, 2009 ) . This is a lipid bilayer construction that differs from cytoplasmatic membrane. This bed non merely made up of phospholipids and protein, but besides polysaccharides that non normally found in cytoplasmatic membrane. Polysaccharide part made up of nucleus polyoses and O-polysaccharides while the lipid part made up of lipoid A which so bind to the nucleus polyoses. This LPS bed is located outside a thin bed of peptidoglycan. The outer membrane gives rises to high lipid composing in the cell wall. Decolourizer dissolve off lipid, hence increases the permeableness of cell wall which finally enables the crystal violet-iodine composite to be lost together with the lipoid.

The cell wall ( murein bed ) of Gram positive bed 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 beds. These acids can be found bonded to muramic acid residues in peptidoglycan. Lipoteichoic acid which refers to the teichoic acids that binds to the lipoids of membrane can besides be found in Gram positive bacterial cell wall. In certain actinobacteria, construction called mycolic acids besides can be found. The deficiency of outer membrane gives rises to low lipid composing in cell wall. Hence, the action of decolorizer on Gram positive bacteriums ( Bacillus sp. , Staphylococcus aureus and Actinomycetes sp. ) causes desiccation of cell wall due to the thick peptidoglycan and the composing of lipid available to be dissolved is low. This finally decreases cell wall permeableness, shuting pores on cell wall and therefore retain the crystal violet-iodine complex interior ( Differential staining: The Gram Stain, n.d. ) . As the cell psychiatrists, the composite trapped in the thick peptidoglycan and hence cells do non decolourized. After this procedure, E. coli is in colourless as the crystal-violet I complex loses while Bacillus sp. , Staphylococcus aureus and Actinomycetes sp. still in violet coloring material.

Ethanol was non added for more than 30 seconds. Over decolourization can do the discoloration of Gram positive bacteriums to bleach and appears as Gram negative ( Betts et al, 2003 ) . Under decolourization ( excessively abruptly ) besides avoided as it can do dye to be removed incompletely from Gram negative bacterium. Both state of affairss can give false consequences. After decolorization, vilification was washed with distilled H2O for 15 2nd to wholly halt the decolourization procedure. The counterstain, safranin solution so stained the E. coli that is colorless with the ruddy coloring material. Safranin is basic dye ( cationic ion ) carry the positive dye ion, chromophore that attached to acidic cell constructions ( negatively charged ) such as the living substance. Basic dye besides attached to other negatively charged supermolecules like proteins and nucleic acid ( Archunan, 2004 ) . Both the Gram positive and Gram negative bacteriums took up the counterstain but the coloring material of Gram Positive do non alter much as it already stained with purple. For every dye, there is different period of clip for staining. This is to forestall over or under discoloration that may consequences in inaccurate consequence.

From the observation, Escherichia coli stained ruddy and give accurate consequence of Gram negative. The form of E. coli can be observed as rod form. Bacillus sp. , Staphylococcus aureus and Actinomycetes sp. while shows consequences of Gram positive as all are stained with violet coloring material. The forms observed are severally bacillar, round-shaped and in mycelial. For Staphylococcus aureus, the coccus form is sticked together in bunchs or formless sheet and non separated. For E. coli, B sp. and staphylococcus aureus, two samples are taken, one from the stock and one from the agar. Both the samples show the same consequences. The difference is on the sum of micro-organisms observed. Bacillus sp. , for illustration, that taken from agar home base is really crowded. This is because the each settlement taken contains a figure of micro-organisms. It is more hard to be observed the form of the beings. However, the coloring material stained can be observed clearly. For the broth civilization, single beings and the form every bit good as the coloring material can be observed more clearly.


Gram staining is of import in distinguishing Gram positive and Gram negative bacteriums in which the Gram positive bacteriums stained violet coloring material while Gram negative beings stained pink. Escherichia coli is Gram negative while B sp. , staphylococci aureus and actinomycetes are Gram positive bacteriums.

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