L7 Lab Staphylococcus and Streptoccus PDF

[LABORATORY] LESSON VII: IDENTIFICATION OF STAPHYLOCOCCUS INTRODUCTION MATERIALS STAPHYLOCOCCUS 1. Bacterial suspension of Staphylococcus aureus. are normally found on skin and mucous...

[LABORATORY] LESSON VII: IDENTIFICATION OF STAPHYLOCOCCUS INTRODUCTION MATERIALS STAPHYLOCOCCUS 1. Bacterial suspension of Staphylococcus aureus. are normally found on skin and mucous 2. Cedarwood Oil membrane as well as in our environment. It is 3. Glass slide difficult to determine whether the given 4. Gram stain reagents staphylococci isolate is pathogenic or not. There to visualize the organisms are no absolute criteria, which distinguish 5. Microscope pathogenic from non-pathogenic strains, but 6. 3% H2O2 (Hydrogen peroxide) pathogenic strains generally hemolyze red blood for the Catalyst Test cells, produce catalase and plasma coagulase 7. 0.5 ml. 1:4 Rabbit's plasma and ferment mannitol. for Coagulate Test Gram (+) cocci 8. Test tubes they are typically found in skin and mucus 9. Cotton swabs. membrane — moist area of the body 10. Wire loops and alcohol lamp they are normal flora — part of normal microbiota of the skin and mucus membrane POST - ANALYTICAL PHASE Staphylococcus Aureus — capable of causing infection because of the toxins PROCEDURE: (especially enzymes) being produced by this 1.) Use cotton swab to collect bacterial sample in particular organisms the skin/nasal cavity of one member of the group. it helps the bacteria established 2.) Using the same cotton swab, inoculate the infection sample in Mannitol Salt Agar. PRE - ANALYTICAL PHASE 3.) Using a sterile wire loop do the step #2 with S. aureus and S. saprophyticus. Sterilization of working area is very necessary 4.) Incubate the plates under 35°C for 18 hours. by using Lysol or bleach to wipe the tabletops. 5.) After incubation, observe for growth and color The laboratory scientist should consider all development. specimen as infectious and must wear personal in order for us to determine kung part ba protective equipment at all times inside the talaga ng normla microbiota yung ating laboratory. Staphylococcus spp. we can collect skin or Prepare Mannitol Salt Agar, Nutrient Agar Blood nasal swab from 1 of the members of the plate. One member will collect skin and nasal group, then inoculate that cotton swab in swabs with other members of the group. your Mannitol Salt Agar of course we need to disinfect your table we can also inoculate S. Aureus and S. your working area and you need to cover Saprophyticus in MSA — so that we can your area compare the colonial morphology of S. Aureus and S. Saprophyticus and the one that will be growing from the skin swab from 1 of the members of your group 1 CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA  TRANS: IDENTIFICATION OF STAPHYLOCOCCUS MANNITOL FERMENTATION YELLOW HALO AROUND COLONIES: POSITIVE (Mannitol fermenter) S. Aureus — we can see yellow colonies and also the agar would also be yellow NO YELLOW HALO AROUND COLONIES: NEGATIVE (Mannitol non–fermenter) other Staphylococcus spp — they produce pink colonies (kakulay lang ng MSA) CELL MORPHOLOGY After incubation observe the color reaction of S. 1.) Prepare a bacterial smear from the bacterial aureus, S. saprophyticus and the skin sample in culture. Always practice aseptic technique. MSA. 2.) Stain with Gram stain contains high concentration of salt 3.) Focus on oil immersion objective High concentration of salt — serve as the 4.) Observe the morphology of the organisms inhibitor for your MSA because some organisms cannot MORPHOLOGY OF STAPHYLOCOCCUS tolerate high concentration of salt. SPP Halophilic Organism — mahihilig sa salt (sila Gram (+) Cocci in irregular clusters lang pwedeng mag grow sa MSA that includes if you focus your gram stain in bacterial your Staphylococcus spp) smear onto your microscope and nakita mo gram positive cocci in irregular clusters magkakabungkos sila (magkaka group sila), and they actually MANNITOL SALT AGAR reassemble bunches of grapes that is characteristic of your contains pH indicator and sugar alcohol Staphylococcus spp mannitol mannitol and ph indicator — gives the MSA it's ability to differentiate the groups of organisms growing into your media one of the clues that hence your MSA is also differential could tell u Staphylococcus spp Among the members of your Staphylococcus ang magde decide spp., it is ONLY S. AUREUS that is able to ng biochemical test ferment mannitol na gagamitin is the — it produce acid by products turning the colonial morphology color of your MSA into YELLOW (colonies) and the gram stain It is very important culture media for the morphology biochemical identification of S. Aureus 2 CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA  TRANS: IDENTIFICATION OF STAPHYLOCOCCUS COLONIAL MORPHOLOGY INTERPRETATION: 1. From the bacterial culture of S. aureus, get an Gas bubbles — POSITIVE inoculum and streak on BAP using the multiple- pag positive sa Catalase Test the bubble interrupted streak method. must be obvious 2. Incubate at 35°C for 18 hours. Pseudo Catalyst Reaction (paisa isa yung 3. Examine the colonies of the organisms. bula then maliliit lang siya) — pseudo 4. Describe the colonies. meaning false (consider negative) Colonial morphology in Blood Agar Plate No gas bubbles — NEGATIVE — your Staphylococcus spp to have PINHEAD COLONIES COAGULASE TEST Pinhead — yung colonies malalaki A. SLIDE OR CLUMPING FACTOR TEST S. Aureus particularly the colony are "golden yellow (with beta hemolysis)" S. Aureus is beta hemolytic — clear zone in agar plate Staphylococcus Aureus — also been describe to have BUTYRI or BUTYROUS COLONIES — the consistency is parang butter BIOCHEMICAL TESTING 1. Place a drop of rabbit's plasma to a clean glass slide. CATALASE TEST 2. Place a drop of saline next to the plasma as control. 3. Emulsify an inoculum of S.aureus in each drop placing first on the saline. 4. Mix well for 5 seconds 5. Observe for clumping within 10 seconds. 6. Record results. important test for identification of S. Aureus — specific for S. Aureus Catalase Test — is for whole genous of Staphylococcus (lahat ng members ng staph 1. Pour 1 ml of a 3% H₂O₂ solution over the 24-hour is positive sa Catalase Test) nutrient agar. Member ng Staphylococcus na nag cause 2. Emulsify a colony using applicator stick in 1 drop ng human disease na positive in Coagulase of 3% H₂O₂ in a glass slide. Test — S. Aureus 3. Observe for the production of gas bubbles. Human isolate na positive in Coagulase differentiate your Staphylococcus which are Test — S. Aureus positive from your Streptococcus which are 2 Methods: Slides Coagulase and Tube negative Coagulase Streptococcus spp — are also gram (+) Slide Coagulase — screening method cocci (bilog na purple) however, the (lahat ng mag negative sa slide coagulase arrangements is mostly in chains or in pairs magpo proceed sa confirmatory test. We are All members of Staphylococcus spp is determining the presence of clumping factor always positive in Catalase Test (also known as bound coagulase) Reagent: 3% H₂O₂ (Hydrogen peroxide) Tube Coagulase — confirmatory method 3 CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA  TRANS: IDENTIFICATION OF STAPHYLOCOCCUS 2 Area of Slide: T for Test (left side, Referred reagent: Rabbit Plasma reagent: 1 drop of Rabbit Plasma) and C for dahil wala naman tayong rabbit, so ang Control (right side, 1 drop NSS) gagamitin natin if we do this would be Control Side (NSS) — whitish milky Human Plasma — extract dugo, ilalagay sa appearance EDTA, mix EDTA and then centrifuge Test Side (Rabbit Plasma) — clumps or agglutinates (positive results) INTERPRETATION: No agglutination in Control Side and no Coagulation formation — POSITIVE agglutination in Test Side (parehong No coagulation formation — NEGATIVE mukhang milky) — Negative Slide Coagulase (proceed in Tube Coagulase) POST ANALYTICAL PHASE INTERPRETATION: After the experiment, all the glassware, Clot formation — POSITIVE materials that have been in contact with bacteria No clot formation — NEGATIVE like inoculating loop and including the working station, should be sterilized properly. The B. TUBE TEST student must prepare 10% bleach or Lysol for disinfection. Materials like inoculating wire can also be utilized by flaming. All disposable materials should be placed properly in correct trash bags. PPEs should be disposed in infectious trash bag and should not be worn outside the laboratory. after testing you now have to clean up, disinfect the table, disinfect the cover then remove your PPEs 1. Inoculate a loopful of S. aureus to 0.5 ml of 1:4 rabbit's plasma 2. Incubate at 35°C for 2 hours then 4 hrs. 3. Observe for coagulation formation. 4. If no coagulation is formed, incubate further up to 24 hours. 5. Observe for coagulation formation after 24 hours, if you see gel like consistency (parang nag solidify gelatin siya) — positive pag liquid padinn yung consistency — you have to incubate it further for another 2 hrs after 4 hrs total incubation, tilt ulit then pag liquid padin incubate it overnight (lagay sa room temp) use to identify S. Aureus — the most pathogenic member of Staphylococcus 4 CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA  TRANS: IDENTIFICATION OF STREPTOCOCCUS AND ENTEROCOCCUS IDENTIFICATION OF STREPTOCOCCUS Some Streptococcus spp are Beta AND ENTEROCOCCUS SPECIES hemolytic — they produce complete hemolysis (blood agar surrounding the Streptococcus and Enterococcus are Gram (+) colonies actually looks clear (destroy cocci also RBC) Streptococcus and Enterococcus usually Some Streptococcus spp are Non appears in chain or by pairs Hemolytic — also known as "Gamma Enterococcus dati part sila ng Strep yun ang hemolysis" they do not destroy the red genous nila dati Strep but they have been blood cells so they are gamma hemolytic reclassified their own genous — kaya nagkaroon na ng Enterococcus PRE ANALYTICAL PHASE Enterococcus are previous members of Strep Sterilization of working area is very necessary by using Lysol or bleach to wipe the tabletops. INTRODUCTION The laboratory scientist should consider all specimen as infectious and must always wear Streptococci are gram positive occurring in personal protective equipment inside the pairs. or in chains, non-motile, facultative Jaboratory. anaerobes and catalase negative organism. again, disinfect the table you need to cover it Some species are well known to cause pyogenic and you need to wear your PPEs infection to man. But some species have been noted to be part of the normal flora that resides in the skin, oral cavity, nasopharynx and genital MATERIALS tract. Streptococci are classified on their hemolytic. Blood agar plate properties; Alpha-hemolysis, causes partial Bacitracin disk (Taxo A) hemolysis that develops a greenish color on 6.5% NaCl blood agar; Beta-hemolysis, causes complete Wire loops hemolysis. that makes the blood agar clear; Petri dish Gamma- hemolysis, there has been no 3% H2O2 (Hydrogen peroxide — for Catalyst hemolysis took place in blood agar test Gram (+) cocci, they appear in chain or Gram stain reagents by pairs depending on the spp. Alcohol lamps Glass slides Some of them can cause human infection — Forceps Cotton swab strep throat, neonatal meningitis, low bar Bacterial broth of the following: pneumonia (Strepto may sala sa mga E. faecalis disease na ito) B-hemolysin-producing strain of S. aureus Streptococcus is also part of normal flora S. pyogenes — makikita sa nasopharynx, sa throat, sa S. agalactiae oral cabity and even genital tract Blood Agar Plate is use for identification of hemolytic patterns Some Streptococcus spp are Alpha hemolytic — they produce partial hemolysis (the media surrounding the colonies appear GREENISH /BROWNISH in color 5 CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA  TRANS: IDENTIFICATION OF STREPTOCOCCUS AND ENTEROCOCCUS ANALYTICAL PHASE Colonies of Streptococci on most enriched media are grayish, translucent to slightly PROCEDURE: opaque, circular, pinpoint colonies. Colonies Cell Morphology may vary from the mucoid or smooth type to the Colonial Morphology matte or rough form. Test for Identification: colonial morphology particularly in BAP Catalase Test — we need to inoculate your specimen in Hemolysis your BAP then observe for hemolytic pattern BACITRACIN DISK TEST Colonies of Strep — pinpoint (maliliit na CAMP TEST colonies) GROWTH ON 6.5% NaCl We can differentiate them base on hemolytic SODIUM HIPPURATE HYDROLYSIS pattern BILE ESCULIN HYDROLYSIS 1. Using cotton swab, collect sample in the throat CELL MORPHOLOGY region of one of the group's member. 2. On a blood agar plate inoculate the swab and the bacterial suspension of other streptococcus organism. 3. After incubation, observe for hemolysis. HEMOLYSIS The Streptococci are Gram-positive cocci normally arranged in chains of varying lengths. Cells are about 0.5um. Streptococcus Pneumoniae — describe to be "lancet shape" and usually appears in pairs A. Beta Hemolytic Streptococcus 1. Prepare a bacterial smear from the bacterial Group A: S. pyogenes suspension of S. pyogenes or S. agalactiae. Always Group B: S. agalactiae practice aseptic technique. Some Group D: Enterococcal and 2. Stain with Gram stain. enterococcal Strep 3. Focus on oil immersion objective B. Alpha Hemolytic Streptococcus 4. Observe the morphology of the organisms S. pneumoniae Viridans Strep COLONIAL MORPHOLOGY Some Group D Streptococcus C. Gamma Hemolytic Streptococcus Some Group D Streptococcus Some Viridans group 6 CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA  TRANS: IDENTIFICATION OF STREPTOCOCCUS AND ENTEROCOCCUS CATALASE TEST Bacitracin Disk Test — actually a type of antimicrobial susceptibility testing (AST) but instead of using Mueller Hinton Agar (MHA), the one that we will be using is Blood Agar Plate (BAP) Bacitracin Disk Test (Taxo A) — contains 0.04 units of antibiotic bacitracin Principle: Your group A (Strep pyogenes) is susceptible to small amounts of bacitracin Problem with Bacitracin Disk Test: It is not specific for Group A only. Some Sample should not be from the blood agar plate members of the Group C may test positive, because it will produce a false positive result. some of them are also susceptible for your This differentiate Staph to your Strep bacitracin or Taxo A (Positive — Staphylococcus; Negative — Better alternative to Taxo A: is your Streptococcus PYR (Pyrrolidonyl Arylamidase) — specific Expecting no bubble presentation for your for Group A strep (Strep pyogenes) Strep (negative sa catalyst test) much better ang PYR in the identification of Strep pyogenes than Bacitracin 1. Emulsify a colony using applicator stick in 1 drop of 3% H₂O, in a glass slide. MATERIALS: 2. Observe for the production of gas bubbles. INTERPRETATION: 1. 18-24-hour broth culture of S. pyogenes. 2. Blood agar plate Gas bubbles — POSITIVE 3. 0.04 units of Bacitracin (TAXO-A) No gas bubbles — NEGATIVE PROCEDURE BACITRACIN DISK TEST 1. Dip a cotton swab in broth suspension; express excessive liquid the swab against. 2. Streak the surface of the BAP. 3. Aseptically apply and incubate for 18 hours at 35°C. 4. Observe for zone of inhibition. INTERPRETATION: Zone of inhibition around disk — POSITIVE No zone of inhibition around disk — Principle: This test is used to differentiate. form NEGATIVE other groups Herentiate groupyte streptococci, based on the sensitivity of S.pyogenes to low concentration of Bacitracin also known as "Taxo A" use to identify your group A strep (Strep A pyogenes 7 CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA  TRANS: IDENTIFICATION OF STREPTOCOCCUS AND ENTEROCOCCUS CAMP TEST Inoculate your S. Aureus vertically, unknown (group B strep) perpendicularly until near the center Why there is a arrow head hemolysis? because this S. Aureus produces beta lysine MATERIALS: 1. Isolated colonies of S. Agalactise on BAP. 2. Beta lysine producing S. Aureus on sheep's blood. Principle: S. agalactiae produces a substance known as CAMP factors which enhances the hemolysis of sheep RBC by Staphylococcal beta PROCEDURE lysine. 1. Inoculate a streak beta-toxin producing S. aureus this is for identification of group B Strep straight on the center of sheep's BAP which is Strep Agalactiae — which causes 2 Inoculate straight line of S. agalactiae at right neonatal meningitis angle to the Staphylococcal streak without touching Group B Strep (Strep Agalactiae) — the colonies of Staphylococcus. known to produce a substance called 3. Incubate at 35°C for 18 hours in a candle jar. "Camp Factor" 4. Observe an arrow-head shaped zone of Camp Test — we need to use a bacterial enhanced hemolysis at the junction of the colonies strain as reagent particularly Streptococcus and Staphylococcus. Staphylococcus Aureus. This Staphylococcus Aureus produces beta lysine (which is cytotoxin) Reagent: S. Aureus that produce beta INTERPRETATION: lysine Presence of arrowhead hemolysis — POSITIVE Camp Test uses BAP (Blood Agar Plate) as No presence of arrowhead hemolysis — media, also Taxo A (BAP) din gamit NEGATIVE The Beta lysine of S. Aureus has a synergistic effect with the Camp Factor produces by S. Agalactiae Synergistic — pagmagkasama sila mas powerful sila (synergism) Concept: Pag magkasama ang Beta lysine produces by S. Aureus and Camp factor produce by S. Agalactiae mas lumalakas ang hemolytic power ng dalawa —magaling sila mag destroy ng RBC They are more effective in destroying the RBC and that what we take advantage of when we are trying to identify S. Agalactiae by determining if camp factor is produced 8 CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA  TRANS: IDENTIFICATION OF STREPTOCOCCUS AND ENTEROCOCCUS MATERIALS: BILE- ESCULIN HYDROLYSIS 1. Isolated colonies on BAP of Group D Streptococcus 2. Bile-esculin agar. PROCEDURE 1. Inoculate one to two colonies on a bile-esculin agar slant or streak in a line on the surface of a bile- esculin agar plate. 2. Incubate at 35°C for 18-24 hours. 3. Observe for blackening of agar slant or blackening around line of growth. INTERPRETATION: Principle: Group D streptococci can grow in the presence of 40% bile and subsequently Blackening of growth or medium — POSITIVE hydrolyze esculin to esculetin and glucose. No blackening of growth or medium — Esculetin diffuses into the agar and combines NEGATIVE with ferric citrate in the medium to give a black complex. GROWTH ON 6.5% NaCl Esculin -------- bacterial enzyme ------> esculetin+glucose Esculetin + Fe² -------> black complex we utilize the bile esculin agar which could be in many forms Bile esculin in plated (nasa petri dish), Bile esculin in slant Color of Bile Esculin: Brown Bile Esculin: test group D 2 general groups of Group D: Group D Enterococci (Entero spp) and Group D Principle: Enterococci can withstand a higher Streptococci (S. Bobis). Both will positive in salt concentration than non-enterococci. Bile Esculin test 6.5% NaCl — is just a nutrient broth but Bile Esculin Agar contains high have a 6.5% NaCl (very salty) concentration of bile (40%) which is tolerated by your Group D MATERIALS: The members of Group D can convert esculin to esculetin — produce black 1. 18-24 hour broth suspension of Enterococci coloration 2. 6.5% NaCl broth Problem: we cannot differentiate which Group D is that PROCEDURE How can we further differentiate kung sino yung tumubo sa bile esculin kung group D 1. Inoculate a loopful of Enterococci to 6.5% NaCl Enterococci or S. Bobis — proceed with 6.5 broth. NaCl 2. Incubate at 35°C for 18 hours. 3. Observe for turbidity. 9 CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA  TRANS: IDENTIFICATION OF STREPTOCOCCUS AND ENTEROCOCCUS INTERPRETATION: When you add ninhydrin reagent it will react the glycine producing a purple color Visible turbidity - POSITIVE Group D Enterococci — they can tolerate MATERIALS: high salt concentration producing turbidity in 6.5% NaCl 1. Isolated colonies of S. Agalactiae. 2. Sodium Hippurate substrate. No visible turbidity - NEGATIVE 3. Ninhydrin reagent. S. Bobis (Group D Streptococci) — they cannot grown in 6.5% NaCl there for they are negative in this particular test PROCEDURE 1. Inoculate a Sodium Hippurate substrate a NOTE: suspension of S. Agalactiae to produce a milky suspension. If it is positive in Bile Esculin (mag grow yung 2. Incubate at 35°C for 2 hours. bacteria) and have a black coloration then if it 3. Add 0.2 ml of Ninhydrin reagent and further is positive in 6.5% NaCl incubate for 15 minutes then that is your Enterococci 4. Observe for appearance of deep purple color. If it is positive in Bile Esculin and have a black coloration but did not grow in 6.5% NaCl INTERPRETATION: then that is your S. Bobis (Group D Formation of deep purple color — POSITIVE Streptococci) No color change — NEGATIVE SODIUM HIPPURATE HYDROLYSIS POST ANALYTICAL PHASE After the experiment, all the glassware, materials that have been in contact with bacteria like inoculating loop and including the working station, should be sterilized properly. The student must prepare 10% bleach or Lysol for disinfection. Materials like inoculating wire can also be utilized by flaming. All disposable materials should be placed properly in correct trash bags. Principle: Based on the presence of enzyme PPEs should be disposed in infectious trash bag hippuricase which is able to hydrolyze hippuric and should not be worn outside the laboratory. acid to glycine and benzoic acid. Glycine when disinfect your area, clean up your work area mixed with ninhydrin reagent is reduced giving a and remove your PPEs color change or purple. this is a test for Group B (S. Agalactiae) S. Agalactiae produce the enzyme hippuricase (also known as hippurate hydrolase) — this can breakdown your hippuric acid, it can destroy it hydrolysis hippuric acid to produce glycine and benzoic acid (by products of enzymatic breakdown) 10 CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA