How To Do Gram Staining

How To Do guanine StainingObservation of micro-organism at a lower place microscope mass be improved by using certain processes and techniques such(prenominal) as the smudgeing. Staining is an beta 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 extra kioskular and intracellular comp peerlessnts of microorganisms can be determined and detected through the injurying. Many microorganisms difficult to be observed under microscope due to their neutral appearance and semitransparent properties as their refractive index almost same as surroundings (Patil et al, 2008). The marque 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 coefficient stain while can be used in distinguishing between different types of microorganisms. Special stain on the opposite 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- confirming and Gram- negative groups through the Gram-staining (Sridhar Rao, n.d.). This process besides important in clinical science laboratory such as to examine and identify bacteria responsible for certain d iseases.Staining process requires the zeal of blot that contains a thin mould of bacteria. The preparation of belittle involves spreading and fixing of microorganisms on the microscope parachute. 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 discolor such as watch crystal chromatic that reacts with acidic component of cell and causes all the bacteria to be stained with the crystal reddish blue or purple. The other soil like the m ethyl radical violet can also be used. The other component, mordant in the Gram stain refers to iodine. Mordant is chemical that increases simile 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 a nonher basic dye that important in giving new colour for cells that decolourized. Counterstain can be the safranin (used in this practical) or the speed of light 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 optimistic 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 rin g 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 ReagentsCrystal violetSolution A Crystal violet 2.0gEthanol, 95% (v/v) 20 mlSolution B Ammonium oxalate 0.8gDistilled water 80 mlSolution A and B mixed.Mordant single 1.0 gPotassium iodide 2.0 gDistilled water 300 mlIodine and potassium blended with mortar, distilled water added during blending until iodine dissolved.Decolorization solventEthanol, 95% (v/v)CounterstainSafranin 0.25 g 2.5 %(w/v)Ethanol 10 ml 9.5% (v/v)Distilled water 90 mlMaterialsGlass seashoreEscherichia coli in broth cultureEscherichia coli in agar culture vitamin B sp. in broth culture atomic number 5 sp. in agar culture staphylococcus aureus in broth cultureActinomycetes sp. in broth cultureActinomycetes sp. in agar cultureKimwipeBunsen burnerDropperDistilled waterInoculation loopProcedurePreparation of smearFor culture taken from liquid metier (br oth), 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.Gram-stainingThe 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 little 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 mic roscope.Results(A)Escherichia coliGDCIM101NIKONDSCN1773.JPG1(a) Broth culture (zoom in).1(b) Agar plate (zoom in).Figure 1 Microscopic image of Escherichia coli under conglomeration effusion of 400- from different culture(B) Bacillus speciesGDCIM101NIKONDSCN1745.JPG GDCIM101NIKONDSCN1738.JPG2(a) Broth culture (zoom in).2(b) Agar plate (zoom in).Figure 2 Microscopic image of Bacillus sp. under total overstatement of 400- from different cultures.(C) Staphylococcus AureusGDCIM101NIKONDSCN1767.JPGFigure 3 Microscopic image of Staphylococcus aureus under total magnification of 400- from broth culture (zoom in).(D) Actinomycetes speciesCUsersmichelleDocumentsUMS MICROBIOLOGYPHOTOSS1.JPG GDCIM101NIKONDSCN1760.JPG4(a) Broth culture (zoom in) under total magnification of 400-.4(b) Agar plate (zoom in) under total magnification of 400-.Figure 3 Microscopic image of Actinomycetes sp. under different magnification from different culture.Table 1 The result of Gram stain on different microorga nismType of microorganismsShape of the microorganismsColour stained on microorganismsGram positive or Gram negativeEscherichia coli (broth culture)Bacillus or Rod-shapedPinkGram negativeEscherichia coli (agar plate)Bacillus or Rod-shapedPinkGram negativeBacillus sp. (broth culture)Bacillus or Rod-shaped olympianGram positiveBacillus sp. (agar plate)Bacillus or Rod-shapedPurpleGram positiveStaphylococcus aureusCoccus or round-shapedPurpleGram positiveActinomycetes sp. (broth culture)MycelialPurpleGram positiveActinomycetes sp. (agar plate)MycelialPurpleGram positiveDiscussionFor 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 mi croorganisms 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 breach 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 sedimentary dye dissociated into CV+ ion and chloride, Cl- ion (Hussey Smith, n.d.). The positively superaerated ion binds to the negatively charged components in cell after penetrating the cell protect 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 th e 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 diminish 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 outmost 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 wish 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 bacte ria. 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 perch shape. Bac illus 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.ConclusionGram staining is important in differentiating Gram positive and Gram negativ e 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.