BIOCHEMICAL TEST TO DISTINGUISH BETWEEN VARIOUS TYPES OF BACTERIA

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1 BIOCHEMICAL TEST TO DISTINGUISH BETWEEN VARIOUS TYPES OF BACTERIA 1. Catalase test : distinguish streptococci from staphylococci (if the catalase test is positive, it will be staph, but if it s negative it will be strep). We do catalase test because sometimes we can't distinguish staph in the gram stain because it look like a cluster. Flood culture with drops of 3% H 2 O 2 hydrogen peroxide " we place a suspension from the needed colony + normal saline on the slide, then we make calcification and add 1 2 drops of 3% H 2 O 2. If the catalase is positive, you can see bubbles on the slide because the catalase positive colony break down H 2 O 2 into H 2 O and O 2 ; these bubbles are due to the release of O 2. ex : S. aureus produces catalase enzymes that convert H 2 O 2 to H 2 O + O 2. Catalase positive: you can see bubbles at once because of released oxygen. What is micrococcaceae (it is written in the slide)??? It's a bacteria similar to staph, which means that it's catalase positive, but it's bigger than staph in gram stain and mostly non pathogenic, unlike staph which is pathogenic. 2. Coagulase: the ability to clot plasma. In staph, we need to differentiate between staph species so we do a test called coagulase test. This test is used to differentiate between S. aureus & other Staphylococcus species; (if the bacteria produce coagulase it's pathogenic which is mostly S.aureus, but if the test is negative it will be other staph species like S. epidermidis and S. saprophyticus). S.epidermidis and S.saprophyticus are both coagulase negative so we need another test to differentiate between them. (We use certain antibiotic because S.epidermidis is sensitive to it, while S.saprophyticus is resistance to) 1 Corrected by: Eman Adel AL Zayadneh

2 Two type of coagulase: very important in the exam 1) Free coagulase: clotting "like JELLY" in tube (the bacteria diffuse and react with proteins) 2) Bound coagulase: slide method. We put serum with a tested microorganism in the slide for 2 min, and see if there is clotting or not. If there is clotting, then the test is positive (the bacteria do not diffuse just remain sticky to the cell). Fibrinogen ( plasma ) Coagulase fibrin (clot ) The tube coagulase test (Free): Procedure: Dr Sameer said: "read the procedure, but you don t have to memorize them". 3. Bile esculin: Mix 0.1 ml of culture ml of plasma Incubate at 37C for 4 h Observing the tube for clot formation Any degree of clotting constitutes a positive test To detect beta glucoside which breaks down esculin, changing the color to black due to presence of ferric ions. We can put it in plate or in tube, but in hospitals, we use plates. We use it to distinguish streptococcus Group "D" and Entereococcus from other microorganism. (If black discoloration occur in the bile esculin media, this means that the bacteria is positive streptococcus group "D" or Entereococci). Streptococcus group "D" and Entereococcus, both are bile esculin positive, so we differentiate between them by "growth in 6 points NACL" Dr. Mohmmad will talk about it later. 2 Corrected by: Eman Adel AL Zayadneh

3 Precipitation is due to presence of ferric ions. 4. Urease Detects urease production: To distinguish between proteus and salmonella because both of them are non lactose fermenters. Procedure: just read them 1. Inoculate a urea tube with 3 loopfuls of slant culture. 2. Incubate 24 hours, observe for reaction. 3. A pink color ( red) formation indicates the breakdown of urea to ammonia and CO 2 Proteus +ve formation of red color in the tube Salmonella ve the urea in the tube remain yallow 5. Oxidase: to test for the production of oxidase. Place a spot of inoculated organism on to a filter paper soaked with 1% reagent called tetramethyl phenylenediamine dihydrochloride positive is dark blue or purple. Negative is yellow. Pseudomonas aeruginosa (+), Neisseria (+), fibriocolera (+) Escherichia coli ( ) 6 Indole test Inoculate the tryptophan broth with the test organism and incubate at 37 C for 24 28h. Add 0.5mL ( 2 3 drops) of the Kovac s reagent Examine the upper layer of liquid Positive result red (ring) colour (occurring within a few seconds) Negative result yellow (ring) colour. Proteus mirabilis : Indole negative (yellow ring ) Proteus vulagary, E.coli : Indole positive (red ring ) 3 Corrected by: Eman Adel AL Zayadneh

4 7 Citrate utilization Streak up the slant with the inoculum. Incubate at 25 or 37 degrees C. Klebsilla in the first 24 hours doesn't form mucoid so how we distinguish it from E.coli??? Using citrate utilization test. Citrobacter, Klebsiella (Positive) blue discoloration E.coli (negative) green discoloration 8 Motility Test: This test is done to help differentiate species of bacteria that are motile. Media: semisolid media. How to Perform Test: Stab motility media with inoculating needle. Reading Results: If bacteria are motile, there will be growth going out away from the stab line, and test is positive and also, if the growth resembled V shape, it s positive. If bacteria is not motile, there will only be growth along the stab line. A colored indicator can be used to make the results easier to see. If the color in the whole tube is dark positive However, if the color appears only in the place you put the needle on negative If the color has spread in the tube space more than the needle space, but the entire tube isn t dark positive. 9 Triple sugar iron agar: a microbiological test named for its ability to test microorganism's ability to ferment sugars and to produce hydrogen sulfide. (Table slide 9) TSI agar medium was developed based on Kligler's iron agar A/A the whole tube become yellow (A for acid) K/K the whole tube is red. 4 Corrected by: Eman Adel AL Zayadneh

5 K/A the slant المائل) (الجزء is red but the rest is yellow. P.S: the table is NOT important 10 MSA "Mannitol salt agar": is a selective differential medium for staphylococci. o The cause of selectivity is due to presence of high salt concentration o The cause of differentiation is because it contains mannitol (sugar) and phenol red (ph indicators turns yellow in acidic ph and turns red in alkaline ph) o Positive: staphylococcus or Micrococcaceae (turn the color from red to yellow). o Negative: the color stay red. 11 API Strips Rapid Tests: (20 test: 10 sugar test and 10 biochemical tests) Commercial biochemical test panels Cover a significant number of clinically important groups of bacteria. Different test panels are prepared in dehydrated forms, which are reconstituted upon use by addition of bacterial suspensions. After incubation, positive test results are scored as a seven digit number (profile). Identity of the bacterium is then easily derived from the database with the relevant cumulative profile codebook. 12 Antibiotics sensitivity testing (test diffusion method or Kirby Bauer method): It is ordered to enable the doctor to choose the most effective Antibiotic against the micro organism with consideration of: a) The case of the patient. b) The age of the patient. Procedure: 1) Take a loop of one colony, which had been previously grown (agar media) by using a sterile swab. 5 Corrected by: Eman Adel AL Zayadneh

6 2) Then by the swab, spread the loop on a nutrient agar media in 3 direction to ensure confluence. 3) antibiotic impregnated disks are placed onto agar surface. 4) Incubate the culture at 37C for 24 hour in an incubator. 5) Observe the results by measuring the diameter of inhibition zone * Every disk has certain amount of antibiotic (30, 20, 10, 5). * We measure the diameter by using certain calibers ( المفتاح اإلنجليزي (زي or by using ruler. * The bigger the zone doesn't mean the more sensitive microorganism to the antibiotic It depends on the diameter that makes the microorganism sensitive, meaning that even if we have a large zone, the microorganism might not be sensitive because the diameter that makes it sensitive is larger than the diameter we got. some important information about gram positive rods (bacilli): listeria (catalase positive ) and motile at 25C bacilli may cause tuberculosis Gram positive cocci Catalase test + Cluster ( coagulase test ) Microcococcacea streptococcus + hemolysis Novobiocon test S. aurus resistance senstive β δ α S. saprophytic S. Epidermis 6 Corrected by: Eman Adel AL Zayadneh

7 β hemolytic : complete hemolysis,clear zone of hemolysis around the colony ( yallow color ) Strep.group A(strep.pyogenus) Strep.group b ( strep. agalactiae) Other β hemolytic strep ( group c,d,f,g, ) Bacitricin ( disk use to distinguish btw β hemolytic streptococci ) sensitive resistance + resistance ( CAMP ) test. Group β streptococci produce extracellular protein (camp factor )act as synergistically with staph β lysin to cause lysis of RBC α hemolytic: partial hemolysis with green discoloration around the colony Strep.pneumonia Strep.viridans δ hemolytic : No lysis Optochin susceptibility test used to differentiate btw different α hemolytic streptococci Sensitive ( do not growth at the presence of optochin) Resistance ( can grow with the presence of optochin ) Bile esculin Grow in 6.5% NaCl Bile esculin 6.5% salt Entrococcigroup d + + Non entrococci group d + Other α hemolytic strep. 7 Corrected by: Eman Adel AL Zayadneh

8 Gram negative bacteria Non fastidious Fastidious( grow in (grow in blood agar with out co2 ) Chocolate agar + 5% of co2 ) Triple suger iron agar k/a A/A K/K Coccibacilli Diplococcic (oxidase positive ) Only Xfactor require (X+V)to growth ( haemoinflunza) Nisseria meningities Nisseria gonorrhae Glucose + glucose + Maltose + maltose K/K (non fermenters), oxidase positive, ex: pseudomonades erogenous, Neisseria, miroxalis K/K (non fermenters), oxidase negative, motile like Acinitobactar A/A Lactose Manntole Indole urea citriate E.coli Klebsiella.pneumonia k.oxytra enterobacter serratia + K/A Shigella(non motile ) +/ K/A with production of H2s salmonella + +/ Proteus mirabilis + (important ) + Proteus velgarus + +( important ) + 8 Corrected by: Eman Adel AL Zayadneh