Lab 1.pdf

‫ﺻدﻗﺔ ﻋن روح اﻟزﻣﯾل أﺣﻣد ﺣﻣودة وﻋن‬ ‫ﻋﺎﻣر ﻧﺎﺻر وﺟﻣﯾﻊ اﻟﻣﺳﻠﻣﯾن و اﻟﻣﺳﻠﻣﺎت‬ ‫ﻓﺎﻟﻠﮭم اﻏﻔر ﻟﻠﻣؤﻣﻧﯾن واﻟﻣؤﻣﻧﺎت واﻟﻣﺳﻠﻣﯾن‬ ‫واﻟﻣﺳﻠﻣﺎت اﻷﺣﯾﺎء ﻣﻧﮭم واﻷﻣوات‬ Blood Culture Pa...

‫ﺻدﻗﺔ ﻋن روح اﻟزﻣﯾل أﺣﻣد ﺣﻣودة وﻋن‬ ‫ﻋﺎﻣر ﻧﺎﺻر وﺟﻣﯾﻊ اﻟﻣﺳﻠﻣﯾن و اﻟﻣﺳﻠﻣﺎت‬ ‫ﻓﺎﻟﻠﮭم اﻏﻔر ﻟﻠﻣؤﻣﻧﯾن واﻟﻣؤﻣﻧﺎت واﻟﻣﺳﻠﻣﯾن‬ ‫واﻟﻣﺳﻠﻣﺎت اﻷﺣﯾﺎء ﻣﻧﮭم واﻷﻣوات‬ Blood Culture Part 1 Prof. Hazem Aqel Blood and Lymphatic System Basic Medical Sciences Al-Balqa Applied University contamination (15 it- Objectives 3,dferer,51s:5dis, "estest ' - WHAT IS BLOOD CULTURE :sesd1:9se: A blood culture is the laboratory test in which blood bottle? 5561ose, ~ septicesses -S. --N is injected into bottles with media to determine the presence of microorganism invaded in blood of - 354 Media (155:28;91 I patient - - S no PURPOSE OF BLOOD CULTURE Antibio,/21 1 no sig DIAGNOSIS - PROGNOSIS - THERAPY - · [?s 45:58382 sc, BACTERAEMIA VS. SEPTICAEMIA -> I The presence of bacteria in the blood is called Bacteraemia Bacteraemia occurs in – v Typhoid fever – Brucellosis ~ – Leptospirosis ~ ~ – Endocarditis * L Severe and life-threateningC form of bacteraemia is called Septicaemia Septic shock is due to Gram negative bacilli like Salmonella  PATHOGENS ISOLATED FROM BLOOD CULTURES Isi BACTERIA FUNGI & se. I' – v E. coli is Jos 95. – S. epidermidis – Candida albicans – Neisseria meningitidis – Yeasts 5S – Salmonella Typhi – S. aureus – Histoplasma w capsulatum 3-4 week: es s 24-48h,s 8.1. s?, NOTE: ini. agarsis & Blood does not have a normal microbial flora. => > jet 7, bac. Day 1 30 highferer 9iss? 1-gig 1.5.1 Collect blood and inoculate culture media -: s jit, gets is6. I Collection of blood: * I-- 2151, s s, - Blood should be collected before antimicrobial Sisi & 3,6. : treatment has started =1652, 5x241; 1 -- · High fever Collect the blood as the temperature of patient begins to rise (fever) 5 · At least two specimens (collected at different times) O should be cultured. (Om/foreach ·I gor.II? bottle About 20 ml of blood is taken from adults. Blood from neonates should be collected from a peripheral vein not from the umbilical vein. Sessio - - 1–2 ml of blood is enough to detect the presence of bacteria in children’s blood.  Choice Of Culture Media - Diphasic blood culture medium ( meriosisis" Columbia agar and broth Commercially produced culture media 8 - main six: ( NOTE: Choice of media depends on the bacterial disease that is · Isd ssd suspected. Some bacteria grow well in a particular medium, others do not. oss, so iss media (1$? Aseptic Blood Collection And Dispensing Technique Sibe! Wash hands. lip' Disinfect the venipuncture site with 70% ethanol and stable 2% tincture of iodine. -> Decontaminate blood culture bottle tops. Using a sterile syringe, withdraw 20ml blood. Dispense 10 ml into the culture medium. Incubate the inoculated media as soon as possible. ✓ Incubate up to 4 weeks when brucellosis is suspected ✓ Incubate up to 14 days for anaerobic infections  ⑪ ③ ⑦ ⑧ Aseptic Technique ⑯ ⑤ ⑬ Examining The Blood Culture -> in day I 1- CENTRIFUGE: a sample of EDTA anticoagulated venous blood or heparinized capillary blood and make smears of the buffy coat layers. 2- STAIN: ⑤9,559= I red blue bacilli(lec.s -ci N 1. = 351-6:e · coxi – Gram smear: for Gram positive and Gram-negative bacteria - > or + detection Bacteris gram stain &is Ith 1"isb ismakite green isZieh-Neelsen ess – Ziehl-Neelsen smear: To detect AFB (acid fast bacteria) - > TB 2 ↳ - 89 – Giemsa or rapid Field’s smear: To detect borreliae, or parasites - ↳ - Icell wall (100 such as trypanosomes, malaria parasites, and microfilariae. -> parasites 18s. ss. · :Ab 9"... S S - 8 8s5 ho - "Is'suffrauene ' 3- DRY the smears, fix with absolute methanol for 2 minutes and stain by the appropriate staining technique.  Blood agan >'j weat C. B, Day 2 And Onwards green 2 d clear zone e Onesier j5 (ie 3s:81$ 3- REPORT ‫ٮيﻦ‬1 ‫ٮﺰ‬/‫ٮﻤ‬#‫ٮﺴ)ٮﻌﻤﻠﻪ ﻟ‬#‫ ﻣ‬macckonkey agar ‫ٮﻮ ال‬#‫ا‬ · 5s ss – Diphasic culture (Columbia agar and broth) lactose frementer and non lactose Check for microbial growth, indicated by colonies ‫ٮﲏ ﻟون زﻫري‬/‫ٮﺔ وٮ)ﻌﻄ‬#‫ٮ‬/‫ٮﺲ اﺣﻂ ﻋ‬1‫ڡ‬# frementer growing on the agar slope, usually beginning at the ‫ٮﻼا ال‬E‫ زي ﻣ‬lactose frementer ‫ٮﺎﻫﺎ ﻫﻮ‬#‫ﻣﻌ‬ agar-broth interface. ‫ٮﻬﺎ‬#‫ٮﺔ وﻣﺎ اﻋﻄ)ٮﲏ ﻟون ﻣﻌ‬#‫ٮ‬/‫ٮﺖ ﻋ‬/‫ٮﺲ ﻟﻮ ﺣﻄ‬1 E.coli – Colonial appearances Colonies of Staphylococci, S. typhi, Brucellae, and Macconkey ‫ زي ال‬non lactose frementer ‫ى‬K‫ه‬ salmonella most coliforms can usually be seen easily, whereas colonies of S. pneumoniae, Neisseria pyogenes, and Y. pestis are less easily species, S. seen. sallmons a &B E. – Pseudomonas and Proteus species produce a film of growth on the coli agar. –When growth is present: – Subculture on blood agar, chocolate agar, and MacConkey agar. aerobic -unaerobi= – Incubate the blood agar and MacConkey agar plates aerobically and the chocolate agar plate in a carbon dioxide atmosphere (candle jar). – Examine a Gram-stained smear of the colonies. 3 – Depending on the bacteria seen, test the colonies further (e.g., for coagulase, catalase, oxidase, urease, and motility). Day 2 And Onwards Large Gram-positive rods Subculture on lactose egg yolk milk agar and incubate - (C. perfringens) anaerobically prtens, salmonellas- s is proters is crease interobacteriaceae (1 motile, urease and - - Subculture the colonies on Kligler - oxidase negative Gram- - iron agar Black color - > -> negative rods are isolated - Salmonela,S) 55 e test H202 - & He,261 5- - positive:bubbles&ls! catalase positive, Gram- - Suspect Brucella species and send H28 on negative coccobacilli are e n for identification. isolated Mark it as ‘High Risk’. Catalase (-)$ interobacteriaceae I) Catalasel) psuedomones) 2.5. SI, 0, 1s is  & 34 2 Examine toluidine blue smear (diphasic culture) Examine thioglycolate culture Subculture and examine microscopically Incubate subculture anaerobically NOTE: When there is no growth, wash slope of diphasic culture. Reincubate cultures and subculture. Contamination Of Blood Culture aseptic technique si is11 is contamination -? si,i Collection of During -, -> blood subculturing continanation $8, 29''s Frequent contaminants include: - Commensal Staphylococci, Micrococci, and - - Diphtheroids - Contaminants from the environment such as species of Bacillus or Acinetobacter.  Other possibilities Occasionally inimmunocompromised patients, organisms usually Occasionally in immunocompromised considered ‘contaminants’ may be pathogenic, especially fungi. patients, organisms usually considered Contamination is indicated when an ‘contaminants’ may be pathogenic, especially organism is recovered from only one fungi. bottle when it should have grown in both thioglycollate broth and the Contamination is indicated when an organism diphasic culture medium or when a mixed microbial flora is isolated. is recovered from only one bottle when it contamination(19955 81 should have grown in both thioglycollate AIDS :859,61361 broth and the diphasic culture medium or 2 - when a mixed microbial flora is isolated. ·"Isis. 5s · ↑ contamination Another Use… Severe and often fatal reactions can be caused by the transfusion of contaminated blood. The bacteriological investigation of a transfusion reaction is as follows:  · is 6: &(182,5115 Appearing unusually dark in color -28 = growth, is! %si ss..81, 6e Visible signs Containing small clots ·* I I, The plasma appearing red, or unusually turbid (examine after centrifuging a sample of the blood). & Wyss 8is.6II growths is! = 37 2,x & Varied At room temperature subculturing At 4°C - otherise Motility Test I NOC1 Tests Gram-stained smear of the plasma Blood Culturing Methods AUTOMATED MANUAL  Automated method - Various types of bottles 25.35 available to isolate a range of organisms i.e., aerobic, anaerobic, Mycobacterium The system detects Carbon Dioxide production This indicates presence of respiring organisms S! WBC ic js False positives can arise from blood samples false positives with high WBC count Manual -'ss, -8.2-  Diphasic Bottle Combination of: -(-O – An agar slope covering one side of the bottle – 40ml of broth Both agar and broth contain growth factors, peptones, yeast extract, hemin, NAD and vitamins Broth contains anticoagulant (SPS: Sodium Polyanethol Sulfonate) 9918e 9 Atmosphere inside bottle is enriched with Carbon Dioxide and maintained at low pressure to create partial vacuum Allows growth of main aerobic microorganisms that commonly cause sepsis Rubber stopper and green screw cap present so easy to directly inoculate bottle using a needle and open positive bottles Using aseptic technique, the broth is * inoculated with 10-12ml of blood and mixed The bottle is tipped to inoculate the agar slope (do not tip blood into top of bottle) soth Bottle is incubated (upright) for 7 days in Diphasic total (4 weeks if Brucellosis is suspected) - I Bottle examined 2-3 times daily for Bottle colonial growth on agar slope, broth turbidity, haemolysis or a deposit Colonies are usually visible where broth is in contact with agar slope If bottle appears negative – tip the bottle to inoculate agar slope again and re- incubate  - 08: Is,is a 8- 0 2S!2 56: 15!!!, facultative i aeroroberrbic , Do dic Me O · turbidity S - - Any Questions?