Name: Kavie TyrellID#: 27120404Course: ClinicalMicrobiology CourseCode: MTCH331Lecturer: Mr.Delbert RobinsonLab#: 1Lab Date 22Jan. 2018LabTitle: Gram Staining Principles of Gram staining:Gram staining is a methodused in microbiology to differentiate between microorganisms and place theminto two distinct groups; gram positive and gram negative bacteria. Thisprocedure involves the staining of the microorganism which will retaincharacteristic color of the stain based on their cell was constituency. It isperformed on body fluids and biopsies when an infection is suspected. The maincomponents of gram stain is a; primary stain (crystal violet), Mordant(Iodine), Decolorizer (ethyl alcohol, acetone), and counterstain (safranin orneutral red). The mordant iodine binds to crystal violet and traps it in thecell. When a decolorizer such asalcohol or acetone is added, it interacts with the lipids of the cell membranecausing the gram negative bacteria cells to lose its out lipopolysaccharidemembrane leaving the inner peptidoglycan layer exposed, while gram positivecells become dehydrated and traps the stain withing the cells due to themultilayered nature of the petodoglycan present.
This step is critical and mustbe done with caution to avoid over decolorizing. Thegram stain will stain gram positive bacteria crystal violet (primary stain) dueto the presence of peptidoglycan in their cell walls, while it will stain gramnegative bacteria red (counterstain) due to thinner peptidoglycan in the cellwalls, therefore they do not retain the crystal violet color. The gram stainingtechnique was named after a Danish bacteriologist Hans Christain Gram(1853-1938) who originally devised it in 1882 to discriminate between pneumococcaland Klebsiella pneumonia bacteria in the lungs. Procedures of Gram Staining:1. Take a clean, grease free slide.2. Prepare the smear of suspension on the clean slide with a loopfulof sample.3.
Air dry and heat fix4. Crystal Violet was poured and kept for about 30 seconds to 1minutes and rinse with water.5. Flood the gram’s iodine for 1 minute and wash with water.6.
Then ,wash with 95% alcohol or acetone for about 10-20 seconds andrinse with water.7. Add safranin for about 1 minute and wash with water.8.
Air dry, Blot dry and Observe under Microscope.Principlesof Novobiocin test:Novobiocintest is used for the treatment of infections due to staphylococci and othersusceptible organisms. Novobiocin is anaminocoumarin antibiotic, produced by the actinomycete Streptomyces nivens,with antibacterial property. It interferes with the unpackaging andrepackaging of DNA during DNA replication and the bacterial cell cycle. Thistest determines whether the bacterium is either susceptible to novobiocin or resistant to thedrug. Susceptible microorganisms are determined by placing a novobiocin-impregnated paper disk ona agarplate that is inoculated with themicrobe that is under investigation.
The microbe is streaked unto the agarmedia covering as much of it as possible, then a novobiocin disk is placed inthe center of the streaked media. As the microbe multiplies to for coloniesduring incubation cells are exposed to the antibiotic diffusing into the agarfrom the paper disk. If the microbes are susceptible to novobiocin, there will be avisible zone of inhibition formingaround the disk, representing an area where the antibiotic concentration hasprevented bacterial growth, and if the microbe be resistant, there will be avisible growth up to the margin around the disk. Procedures of Novobiocin test:1.
Allow containers to cometo room temperature before use.2. Using a pure 18-24 hourculture, prepare a suspension of the organism; equivalent to a McFarland 0.5opacity standard; to be identified in Tryptic Soy Broth , Sterile Water, or Brain Heart Infusion (BHI) broth3. Inoculate Mueller Hinton Agar, 5% Blood Agar, or Tryptic Soy agarplate with a sterile swab to obtain confluent growth.
4. Aseptically apply one 5ugnovobiocin disk onto the inoculated agar surface and lightly press down toensure full contact with the medium.5. Incubate plate aerobically for18 to 24 hours at 35 to 37°C.6.
Measure (in millimeters)the diameter of the zone of inhibition around the novobiocin disk, and recordas susceptible or resistant.Principles of Catalase Test:Catalase is an enzyme producedby bacteria that respire using oxygen, it catalyses the reduction of hydrogenperoxide. The test is performed to differentiate between catalase-positivestaphylococci and catalase-negative streptococci bacteria. The enzyme initiatesthe breakdown of hydrogen peroxide into oxygen and water as shown in the equationIn routine culture 3% H2o2is used, while in 15% H2o2 is used for the detection ofcatalase in anaerobes. Catalase-positive bacteria are determined when small inoculumsare introduced into hydrogen peroxide and rapid effervescence of oxygen bubblesoccurs. Catalase-negative bacteria will show a lack/weak oxygen bubblesproduction when introduce to hydrogen peroxide. Catalase test is also used to differentiate aerotolerant strainsof Clostridium, whichare catalase negative, from Bacillus species, which arepositive, it can be used as an aid to the identification of Enterobacteriaceae,valuable in differentiating aerobic and obligate anaerobic bacteria, also usedfor the identification of Mycobacterium tuberculosis.Procedures: The test can be done bytwo methods.
a) Slant methodb) Slide method a) Slant Method:1. Using a steriletechnique, inoculate each experimental organism into its appropriately labeledtube by means of a streak inoculation.2. Incubate all culturesfor 24-48 hours at 37?C.3. Allow three or fourdrops of the 3% hydrogen peroxide to flow over the entire surface of each slantculture.
4. Examine each culture forthe presence or absence of bubbling or foaming. b) Slide Method:1. Divide a clean glassslide into two sections with grease pencil.
One should be labeled as “test” andthe other as “control”.2. Place a small drop ofnormal saline on each area.
3. With a sterilized andcooled inoculating loop, pick up a small amount of the culture from thenutrient agar slant or Petri plate.4. Emulsify one or twocolonies on each drop to make a smooth suspension. The smear should be aboutthe size of a pea.5.
With a Pasteur pipette,place one drop of hydrogen peroxide over the test smear. Be careful not to runthe drops together.6.
Do not put anything inthe other drop that serves as control.7. Observe the fluid overthe smears for the appearance of gas bubbles.8. Discard the slide in ajar of disinfectant. Principles of Coagulase Test:Coagulase is an enzyme produced by S. aureus that converts(soluble) fibrinogen in plasma to (insoluble) fibrin.
The free coagulase secreted by S.aureusreacts with coagulase reactingfactor (CRF) in plasma to form a complex, which is thrombin. This convertsfibrinogen to fibrin resulting in clotting of plasma. It can be producedin two forms; bound and free coagulase. A coagulase test is performed in theefforts to differentiate coagulase positive Staphylococcus aureus bacteria fromcoagulase negative bacteria Staphylococcus. Free coagulase is an extracellularenzyme that can be detected in tube coagulase test while bound coagulase is acell wall associated protein that can be detected in slide coagulase test.According to an article from Microbiology and infectious disease; Boundcoagulase is also known as clumping factor.
It cross-links ? and ? chain offibrinogen in plasma to form fibrin clot that gets deposited on the cell wallof the cocci. As a result, individual coccus sticks to each other and clumpingcan be observed. Thefree coagulase secreted by S. aureusreacts with coagulase reactingfactor (CRF) present in plasma to form a complex, thrombin. This convertsfibrinogen to fibrin resulting in clotting of plasma.Procedures:The enzyme coagulase isdemonstrated invitro by two methodsa) The Slide coagulase testb) The Tube coagulase testa) The Slide coagulase test1.
Divide the slide intotwo sections with grease pencil. One should be labeled as “test” and the otheras “control2. 2Place a small drop ofdistilled water on each area3. Emulsify one or twocolonies of Staphylococcus on blood agar plate on each drop to make a smoothsuspension4. The test suspension is treatedwith a drop of citrated plasma and mixed well with a needle5.
Do not put anything inthe other drop that serves as control. The control suspension serves to ruleout false positivity due to auto agglutination6. Clumping of cocci within5-10 seconds is taken as positive. Some strains ofS.aureus may not produce bound coagulase, and such strains must be identifiedby tube coagulase test.b) The Tube Coagulase Test1.
Three test tubes aretaken and labeled “test”, “negative control” and “positive control”.2. Each tube is filled with1 ml of 1 in 10 diluted rabbit plasma.3.
To the tube labeledtest, 0.2 ml of overnight broth culture of test4. bacteria is added.
5. To the tube labeledpositive control, 0.2 ml of overnight broth culture of known S.aureus is added6. To the tube labelednegative control, 0.2ml of sterile broth is added.
7. All the tubes areincubated at 37oC and observe the suspensions at half hourly intervals for aperiod of four hours.8. Positive result isindicated by gelling of the plasma, which remains in place even after invertingthe tube.9. If the test remainsnegative until four hours at 37oC, the tube is kept at roomtemperature for overnight-incubation. Hemolysis:Heamolysis in microbiologyis the lysing of red blood cells due to the bacterial protein known ashemolysin which result in the release of hemoglobin from the RBCs. It isobserved in blood agar to determine the different bacteria that produces theseenzymes.
The degree at which hemolysis occur during bacteria culture helps todistinguish between the different types of bacteria. The three types of heamolysisthat can be observed are Beta, Gama, and Alpha hemolysis. According toencyclopedia.com; “Alpha hemolysis is a greenishdiscoloration that surrounds a bacterial colony growing on the agar. This type of hemolysis representsa partial decomposition of the hemoglobin of the red blood cells. Alphahemolysis is characteristic of Streptococcuspneumonia and so can be used as a diagnostic feature in theidentification of the bacterial strain. Beta hemolysis represents a completebreakdown of the hemoglobin of the red blood cells in the vicinity of abacterial colony. There is a clearing of the agar around a colony.
Betahemolysis is characteristic of Streptococcuspyogenes and some strains of Staphylococcusaureus. The third hemolysis Gamma hemolysis is a lack of hemolysis inthe area around a bacterial colony. A blood agar plate displaying gammahemolysis actually appears brownish. Gamma hemolysis is a characteristicof Enterococcus faecalis”. Results:ColonialMorphologies (Table 1) Morphologies S. aureus S. epidermidis S. saprophyticus Hemolysis Yes No No Size Small Small Small Color Cream Cream White Transparency Opaque Opaque Opaque Shape Entire Entire Entire Consistency Moist Moist Moist Topography Raised Raised Flat Gram Stain,Coagulase and Catalase Test (Table 2) Bacteria Coagulase Test Catalse Test Gram Stain S.
aureus Positive Positive Gram Positive C. S. epidermidis Negative Positive Gram Positive C. S. saprophyticus Negative Positive Gram Positive C. Novobiocin Test(Table 3) S.
aureus Susceptible S. epidermidis Susceptible S. saprophyticus Resistant Discussion:Quadrantstreaking was done on blood agar plate to culture S.aureus, S.epidermidis, andS.saprophyticus bacteria which was then incubated and stained using the gramstaining method. After incubation the colonial morphologies of each bacteriawas recorded as shown in Table 1 of the result which also shows thats.
epidermidis and s.saprophyticus where negative for hemolysis, while s.aureuswas positive for beta hemolysis. Beta hemolysis is describes as the straw halothat appears around bacteria such as s.aureus.
It isa complete lysis of red cells in the media around and under the colonies.Streptolysin, an exotoxin, is the enzyme produced by the bacteria which causesthe complete lysis of red blood cells. The Gram stain of themicroorganisms method was carried out by first selecting a slide, label anddate it with necessary information such as name of microbe and initials, afterwhich a sterile loop was used to place a drop of water onto the slide, the wasthen sterilize and used to touch a colony of the cultured bacteria that was incubatedand suspended in the drop of water on the slide, the slide was left to air dryafter which it was heat fixed and stained. After staining the slide is viewunder oil immersion and it was determined that s.aureus, s.saprophyticus ands.
epidermidis was GPCs due to their crystal violet appearance observed underthe microscope. Catalase and Coagulase test was also performed on the microbesand the results that were obtained showed that S.aureus was positive forcatalase and coagulase, S. epidemidis and s. saprophyticus was negative forcoagulase and positive for catalase test. The coagulase test was carried out byinoculating there plasma tubes with each of the bacteria and incubating thetubes at 370c.
After incubation it was observed that S.aureus was coagulasepositive, while S. epidermidis and S. saprophyticus were coagulase negative asshown in Table 2 of the results. The three bacteria were test againstnovobiocin to determine if they are susceptible or resistant against the drugand it was confirmed that S.areus and S.epidermidis were susceptible andS.saporphyticus was resistant.
The test was performed by inoculated a biplateagar via streaking (which covers the entire agar section in contrast toquadrant streaking) with the different bacteria and placing a novobiocin diskin the center of each sections of the biplate. The inoculated biplate were thenincubated and observed to determine the growth of each bacterium around thenovobiocin disk. Susceptible microbe’s growth shows a clear bacteria free zonearound the novobiocin disk which give a suggestive conclusion. The zone ismeasured to confirm susceptibility and the zone should be greater than or equalto 14mm to confirm susceptibility while a less that 14mm would indicate thatthe microbe is resistant to the drug. Resistant microbes grow all the way up tothe novobicin disk which shows that the drum does not have any effect againstthe bacteria as was observed with S.saprophyticus.
Conclusion:S.epidermidis,S.aureus, and S.saprophyticus was identified. References:· “Gram Staining.
” Microscopy, 3 Nov.2016, serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html.· “Gram stain.
” Medical microbiology,medimicro.blogspot.com/2008/10/gram-stain.html.
https://microbiologyinfo.com/gram-staining-principle-procedure-interpretation-examples-and-animation/· Catalase Test. (n.d.). Retrieved January22, 2018, from http://vlab.amrita.
edu/?sub=3&brch=73&sim=703&cnt=2· Blood Agar, Hemolysis, and Hemolytic Reactions.(n.d.).
Retrieved January 23, 2018, from http://www.encyclopedia.com/science/encyclopedias-almanacs-transcripts-and-maps/blood-agar-hemolysis-and-hemolytic-reactionsBloodAgar, Hemolysis, and Hemolytic Reactions. (n.
d.). Retrieved January 23, 2018,from http://www.
encyclopedia.com/science/encyclopedias-almanacs-transcripts-and-maps/blood-agar-hemolysis-and-hemolytic-reactions· Aryal, S. (2017, May 08). Catalase Test-Principle, Uses, Procedure, Result Interpretation with Precautions. RetrievedJanuary 23, 2018, fromhttps://microbiologyinfo.com/catalase-test-principle-uses-procedure-result-interpretation-with-precautions/