How To Do Gram Staining

How To Do universal gravitational constant spyingObservation of microorganism infra microscope open fire be improved by victimisation real dishes and techniques such as the cracking. Staining is an grave graduation to observe microorganisms more clearly, to differentiate between microorganisms as tumefy as to differentiate parts in microorganism (Bagyaraj et al, 2005). The acknowledgment, morphology, somewhat extra carrellular and intra boothular components of microorganisms back be determined and detected by the staining. Many microorganisms difficult to be discover nether microscope due to their pale appearance and semitranspargonnt properties as their refractive index almost homogeneous as surroundings (Patil et al, 2008). The stain improves contrast for visualizing microorganisms. Staining process push aside be explained either as physical, chemical answer or conspiracy of the both(prenominal) reaction.There argon different types of staining such as the s imple stain, differential stain and special stain. Simple stain screw be used for observing indisputable basic structures as easily as the shape of microorganisms. Differential stain bandage toilette be used in distinguishing between different types of microorganisms. Special stain on the early(a) hand can be used for identifying specific structures in the microorganisms such as the flagella (Frey Price, 2003). gravitational constant-stain is one of the normally used differential stains. The constant of gravitation-staining process discovered in 1882 (published 1884) by Hans Christian thou, a Danish bacteriologist and plays an important role in the classifying the bacterium. gee-staining is usually the initial step in identification bacterium and can be used in characterizing bacteria. Bacteria species can be separated into twain large groups, which are the constant of gravitation- haughty and disconfirming groups through the Gram-staining (Sridhar Rao, n.d.). This process too important in clinical lab such as to examine and identify bacteria responsible for certain diseases.Staining process requires the preparation of touch that contains a thin floor of bacteria. The preparation of smear involves splaying 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 principal(a) stain, mordant, de color inizing agent as well as the counterstain that used in sequences. The prime stains usually basic dishonor such as vitreous silica over-embellished that reacts with venomousic component of cell and powers all the bacteria to be blot with the crystal imperial or purple. The other soil the analogouss of the methyl radical purplish can also be used. The other component, mordant in the Gram stain refers to iodine. Mordant is chemical that increas es relationship 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 dye (Rajan, 2005). The degree of decolorization different in bacteria depends on their chemical components. De wringization agent commonly refers to ethanol or other tooth root like acetone or mixture of acetone and ethyl alcohol. Counterstain while is a nonher basic dye that important in endowment new colour for cells that decolourized. Counterstain can be the safranin (used in this practical) or the carbon fuchsin.The Gram stain (differential stains) overhauls different colour for different types of bacteria. The colour is the one that determine whether the bacterium is Gram confirmatory or Gram negative. The Gram imperative bacteria resist decolourization and give result of crystal violet or purple colour (primary stain). Gram-negative bacteria decolorize an d 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 smother 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 quartz glass violetSolution A vitreous silica violet 2.0gEthanol, 95% (v/v) 20 mlSolution B Ammonium oxalate 0.8gDistilled body of water supply 80 mlSolution A and B mixed.Mordanttincture of iodine 1.0 g kibibyte iodide 2.0 gDistilled water 300 mlIodine and potassium commix with mortar, distilled water added during blending until iodine fade out.Decolorization solventEthanol, 95% (v/v)CounterstainSafranin 0.25 g 2.5 %(w/v)Ethanol 10 ml 9.5% (v/v)Distilled water 90 mlMaterials deoxyephedrine slideEscherichia coli in ancestry agri kitchen-gardeningEscherichia coli in agar-agar gardeningBacillus sp. in broth cultureBacillus sp. in agar cultureStaphyloc occus aureus in broth cultureActinomycetes sp. in broth cultureActinomycetes sp. in agar cultureKimwipeBunsen burnerDropperDistilled waterInoculation loopProcedurePreparation of smearFor 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 card smear. For culture taken from solid medium (agar), one drop of distilled water stolon dispensed on the slide. The single colony then spread on the water to form circular smear.The slide was passionateness-fixed with flame.Gram-stainingThe slide was fixed 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 base 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 dis tilled 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.Results(A)Escherichia coliGDCIM101NIKONDSCN1773.JPG1(a) Broth culture (zoom in).1(b) Agar main office (zoom in).Figure 1 Microscopic scope of Escherichia coli under total ebullition of cd- 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 exaggeration of cd- 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 magnificatio n 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 microorganismType of microorganismsShape of the microorganismsColour varnished on microorganismsGram positive or Gram negativeEscherichia coli (broth culture)Bacillus or baculiformPinkGram negativeEscherichia coli (agar plate)Bacillus or rodlikePinkGram negativeBacillus sp. (broth culture)Bacillus or baculiform loftyGram 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 through without washing away bacteria together with stain. The spreading process (for both brot h and agar culture) enables the distribution of bacteria on slides so that suitable density of bacteria can be tack on the slide. This increases lot 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 waken fixed after drying. Heating enables coagulation and precipitation of protein of bacteria to occurs, consequently 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 non be placed directly above the heat or passed through too many times as heat may causes changes in the shape and therefrom cause the distortion of the mic roorganisms. At the selfsame(prenominal) 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 bearing 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 smother and cell membrane, hence giving the purple colour. The extra crystal violet dye that not binds to cell is clea red by distilled water. Addition of iodine in next step enables the crystal violet dye to further fix and adhere to organisms (Medical facts of life 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 lipoide to be extracted or dissolved from the cell beleaguer 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 away(p) 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 confounded 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 tei choic 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. raw material 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. deductionGram 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.