TRACING THE CRIMINAL Part fourteen: Repeating, or How to get prepared for the practical exam Institute of Microbiology shows L Organisation of practical exam nUsually student picks one of 50 tasks nOne topic of practical sessions corresponds to 2 to 4 tasks; some tasks are related to more than one practical sessions (e. g. ASO – related to both neutralisation and streptococci n Some tasks are mostly practical (like Gram staining), some are rather discussion with practical parts J01+02 Microscopy nThree tasks nWet mount – for large and/or motile microbes (parasites, fungi, motile bacteria) nTo know also: Dark field wet mount (mainly spirochets) nGram staining – how to do it, + survey of other staining methods (Giemsa, Ziehl Neelsen, Burri…) nObservation of already stained preparations – mainly interpretation Wet mount – do it practically nDo not forget to cover the preparation by a coverslip and to use non immersion objectives, magnifying e. g. 4×, 10× or 40×. nWe use no immersion oil nAfter having it done, observe the objects in the microscope Wet mount – procedure Nativní preparát Stained preparation Jednoduché barvení Gram, Lugol, Alcohol, Safranin Tap water use Drying with filtration paper Gram staining – principle Chemical Gram-positive Gram-negative Crystaline violet Staining violet Staining violet Lugol iodine Confirmation Less confirm. Alkohol Not decolorized Decolorized Safranin Remain violet Stain to red Gram non staining bacteria do not stain in the first step, because of lack of any cell wall (Mycoplasma) or a very hydrophobic type of the cell wall (Mycobacterium). Spirochetes would stain gram-negative, but they are very thin, so they, too, use to be often considered to be „Gram non-staining“ and Gram staining is not used in diagnostic. Gram staining – procedure nGentian/crystaline violet = Sol. Gram-Nowy (20 –) 30 sec. nLugol (20 –) 30 sec. nAlkohol 15 (– 20) sec. nrinse by tap water!!! imporant! nSafranin 60 – 120 sec. nrinse by tap water ndry by filtration paper nmicroscopy as in Task One Dift1 P1010003 Specimen microscopy Strain microscopy Foto O. Z. In observation of slides… n…it is important to have basic knowledge of size and morphology (yeast × staphylococci etc.) and to know something about interpretation (= knowledge of clinical microbiology, not just J01!) nFor example: nWBCs = inflamation nno WBCs in sputum = not properly taken specimen nG- diplococci inside WBC, urethral swab – suspicion for gonorrhoea J03 – Culture. Two tasks: Reinoculate a strain Rozočkování Sterilize your loop Take the strain Inoculate first phase Sterilize your loop Do not take the strain again Inoculate second phase Sterilize your loop Do not take the strain again Inoculate third phase Sterilize your loop Do not take the strain again Inoculate the „serpent“ crossing previous lines! One more: Observe the media n1. Broth n2. VL-broth n3. selenite broth n4. Sabouraud n5. Löwenstein-Jenssen n6. Blood agar n7. Endo agar n8. MH n9. 10 % NaCl n10. VLA n11. XLD (+ MAL) n12. CHA n13. Levinthal n14. Slanetz-Bartley Some more media at special bacteriology tasks. J04 – biochemical identification tests One only task, but other stuff is in other topics! nIn special bacteriology, you might meet: nCatalase test nTests with diagnostic strips (oxidase, PYR, INAC) nHajna medium (red = G-NF, other = ENT/VIB) nEventually also MIU (not necessary to know details) nThe only „pure biochemical“ task nENTEROtest 16 or something simillar (STAPHYtest etc.) Do not forget the ONPG test it should be read before the other tests 1 2 H 3G 4 F 5 E 6 D 7 C 8 B 9 A 10 H 11 G 12 F 13 E 14 D 15 C 16 B 17 A First row of the plate 2nd row of the plate + S l l l l l l l l l l l l l l l l – S l l l l l l l l l l l l l l l l ? S l l l l l l l l l l l l l l l l ? + – + + + – – – – – – – – + + + + 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 5 3 0 0 6 3 Decontamination tests – J05 nImportant basic information: nIf we want to find survival limit of bacteria, we have to remove the tested extreme parameters to the conditions and to let them then in optimal conditions for a sufficient time. nIn testing of disinfection effect, bacteria are treated by a disinfectant and then cultured on a medium without disinfectant nIn testing of sterilisation, bacteria are placed to the sterilizer and then cultured in normal conditions Microbes and outer influences Sometimes the action of factor combines The factor allways important is the time A resistant, spore forming bacterium 160 °C 170 °C 180 °C 20 min survives survives dies 30 min survives dies dies 60 min dies dies dies Plus (to both tasks): nAn extra sub-task to both tasks: Make pairs of cards with names of methods/disinfectants and cards of characterisation of methods nThe cards are on a working table, it is possible to see it J06: Atb susceptibility Diffusion disc test: to read it, to interprete it atbpsae21 www.medmicro.info Microdilution test – reading nIn case of collumns 1, 3, 4 & 5, MIC > the highest value nIn wells 8 and 11, MIC £ the lowest value C:\Documents and Settings\u Svate Anny\Dokumenty\Obrázky\Antibiotické testování\P3160025ux.JPG For interpretation, comparison with breakpoints is necessary E-tests – reading nWe can read the MIC value directly on the strip – in place, where the margins cross the strip etest etest www.uniklinik-ulm.de Beta-lactamases detection: limited knowledge is sufficient C:\Uživatel\Ondra\Obrázky a fotky\Odborné\Focené\ESBL\I395.jpg C:\Uživatel\Ondra\Obrázky a fotky\Odborné\Focené\ESBL\I395a.jpg It is not directly in the tasks, but the examinator may ask you about it. Photo O. Z. J07–J09 (but also related with many others): serology Antigen detection: laboratory (animal origin) antibodies + pacient‘s sample or microbial strain. Direct method Antibody detection: laboratory antigen (microbial) + pacient‘s serum (or saliva). Indirect method Antigen a protilátka II Antigen a protilátka I Interpretation nAntigen detection (including antigen analysis): it is a direct method. Positive result means presence of the microbe in the pacient‘s body nAntibody detection: it is an indirect method. Nevertheless, there are some ways how to get the information – when the microbe met the body: nAmount of antibodies (relative – titre + titre dynamics; agglutination, CFT, neutralisation) nClass of antibodies: IgM/IgG (reactions with labelled components – mostly ELISA and immunoblotting) n(Avidity of antibodies) Precipitation Precipitace Two examples of precipitation n1) Microprecipitation in agar according to Ouchterlony ouchterlony + - - - 2) RRR/RPR reaction for syphilis diagnostics (flocculation) Agglutination Aglutinace Agglutination for antigen detection or antigen analysis nEPEC detection: in what situation we perform it, how it is performed… (Practically, you obtain a strain and you have to know what to do with it) nCSF agglutination (task „Comment a videoclip“). Important: besides microscopy this is the second way what to do as quick diagnostics of purulent meningitis Agglutination reaction for detection of antibodies nK+ positive, titre = 1 : 200 nNo 1 negative nNo 2 posit., titre = 1 : 400 nNo 3 negative nNo 4 posit., titre = 1 : 200 1:100 1:200 1:400 1:800 Aglutination on carriers Aglutinace na nosičích Example MHA-TP (www.medmicro.info) TPHA detail +++ ++ + +/- - - - - CFT – principle C:\Uživatel\Ondra\Pracovní věci\Výuka\Výukové materiály\Medici\KFR.JPG Anticomplementarity test C:\Uživatel\Ondra\Pracovní věci\Výuka\Výukové materiály\Medici\KFR antikomplementarita.JPG this is not so important Neutralisation schematically nAntibody (Ig) prevents an effect of a toxin/virus to a cell / red blood cell C:\Uživatel\Ondra\Obrázky a fotky\Odborné\Kreslené\Červená Karkulka bez protilátky.bmp C:\Uživatel\Ondra\Obrázky a fotky\Odborné\Kreslené\Červená Karkulka s protilátkou.bmp Cell in a tissue culture or a red blood cell Toxin or virus Toxin or virus Antibody + – Cell in a tissue culture or a red blood cell Examples of neutralisation reactions Neutralised Object Reaction Bacterial toxin (haemolysin) RBC haemolysis ASO Virus RBC agglutination HIT Virus Cell metabolic effect VNT Important: What is the antistreptolyzin O and why we attempt to detect it nAfter every streptococcal infection antibodies are produced, often including antibodies against streptococcal toxin – streptolysin O. nNevertheless, sometimes after infection the antibodies increase instead of decreasing. Antibodies are bound to some structures of the host organism (autoimmunity), so a „circulus vitiosus“ starts to run nIn such a situation, paradoxically the antibodies are worse than the pathogen that challenged the antibody response to protect us. HIT nHaemagglutination Inhibition Test: Pay attention, it is NOT an agglutination reaction, it is a neutralisation! Antibody neutralises the aggregation of RBCs due to viruses. nSo: Potato-like shape = negative response. Dense round target = positive response nHIT differs from ASO reaction mostly by the fact, that the RBCs are not haemolyzed, but agglutination. But the fact, that a specific antibody blocates the reaction is valid in both of the Reactions with labelled components Laboratory antibody Laboratory antibody Searched antigen Antigen missing Labelled laboratory antibody (àdetection) Labelled laboratory antibody + – It is not bound it is washed away it cannot be detected SURFACE (slide, bottom of a well in a serological panel) Patient specimen ELISA – an example (www.medmicro.info) ELISA pro průkaz protilátek. Klikni! ELISA reaction negative well positive well One task: HBsAg / anti-HBS puzzle nHBsAg testing – positive nHBsAg testing – negative nanti-HBs testing – positive nanti-HBs testing – negative n Reading of ELISA BL 4 BL 4 K- 5 K- 5 K- 6 K- 6 K+ 7 K+ 7 K+ 8 K+ 8 1 9 1 9 2 10 2 10 3 11 3 11 c. o. (IgA) = (0.107 + 0.137)/2 + 0.320 c. o. (IgA) = 0.122 + 0.320 = 0.442 90 % c. o. = 0.398 110 % c. o. = 0.486 all values bellow 0.398 are negative all values above 0.486 are positive c. o. (IgG) = (0.034 + 0.029)/2 + 0.320 c. o. (IgG) = 0.032 + 0.320 = 0.352 90 % c. o. = 0.317 110 % c. o. = 0.387 all values below 0.317 are negative all values above 0.387 are positive FIND POSITIVE AND BORDERLINE WELLS FOR BOTH IgA and IgG! IgA IgG Western blotting – principle n1: original antigen (mixed) n2: decomposition of antigen by a detergent n3: electroforetic division of antigen n4: „blotting“ of divided antigen to a nitrocelulose membrane n5: ELISA reaction (only some antibodies present) C:\Uživatel\Ondra\Obrázky a fotky\Odborné\Kreslené\Western blotting.bmp Western blot – example (picture from www.medmicro.info) Western blot. Klikni! Immunochromatography + – Test area Control area Important! nIt is necessary to know not only how to read the test, but also how to interprete it. nThere are two special tasks (concerning toxoplasmosis and Lyme disease) based on complex interpretation of all results including anamnesis! nE. g. pregnant woman with IgG anti-toxoplasmosis is NOT ill, but protected! J10: DNA detection (PCR) Mostly use of PCR in medical microbiology nThe methods are used mostly in situations, where microscopic and culture diagnostic is difficult or impossible nIt is not very useful for common, ubiquitous pathogens. Because of its sensitivity they would detect accidental molecules comming from environment nThe methods are neither useless, as some people think, nor all-problems-solving, as some other people suppose. Survey of interpretation Proper reaction Internal control Interpretation negative positive negative negative negative inhibition of reaction positive positive positive positive negative (highly) positive C:\Documents and Settings\u Svate Anny\Dokumenty\Obrázky\pcrtbc upravený.JPG An expample of a gel Patients 1 and 4 – positive, patient 2 – negative, patient 3 – inhibition of reaction. 5 – positive control, 6 – negative control, 7 – ladder proper reactions internal controls J11+12: viruses nMajority of viral tasks are serological examinations (HBsAg, anti-HBs) nTwo extra virological tasks concern isolation of viruses nFertilized egg – parts of fertilised egg, used for isolation nCytopathic effect – what is it, how to find it Fertilized egg (+ how to get the info that virus is there) C:\Documents and Settings\u Svate Anny\Dokumenty\Obrázky\Vajíčko\01 Schéma vajíčka.gif AM – amniotic sac, YS – yolk sac, AL – allantois CH – chorioallantoic membrane (CAM) SH – shell membrane (paper membrane) AB – albumen http://www.scielo.cl/fbpe/img/bres/v38n4/fig02.gif C:\Uživatel\Ondra\Obrázky a fotky\Odborné\Z internetu\Medici praktická zkouška\Úkol 42\01 slide05.jpg C:\Uživatel\Ondra\Obrázky a fotky\Odborné\Z internetu\Medici praktická zkouška\Úkol 42\10 MLEC.jpg www.herpesdiagnosis.com/diagnose.html http://cmir.mgh.harvard.edu/cellbio/cellculture.php?menuID_=122 (HSV is Herpes Simplex virus – HSV 1 causing mostly herpes labialis, HSV 2 herpes genitalis) No CPE CPE present J13 Parasitology nAs a basis, we use methods based on modified wet mount: nIn Kato method counterstain with malachite green is used, to make parasites better visible nFaust method is a concentration one (see later) nGraham method is used in pinworms only (and as one task you can do it practically!) nWet mount „sensu stricto“ and stainded preparations (e. g. trichrom) are used in increased suspicion for intestinal protozoa (either primarilly, or after seeing Faust and Kato) Morphology of eggs of intestinal parasites nYou should know at least these shapes to the examination – another task eggs Škrkavka Tenkohlavec bičíkový taenia1 Pinworm Enterobius Roundworm Ascaris Trichuris Tapeworm Taenia Pictures taken from CD-ROM „Parasite-Tutor“ – Department of Laboratory Medicine, University of Washington, Seatle, WA Toxoplasmosis – another task, including definition of patients nP: healthy pregnant woman, cats at home nQ: another healthy pregnant woman, no cats nR: a young lady trekking in forest; no cats, but contact with objects comntaminated by faeces of wild animals nS: a senior, working in garden, cats use to walk throught the garden, symptomas of retinitis + enlarged lymphonodes P01 to P06, P10 nThere is a universal task to those topics: n„Among given strains, find strain(s) of Xxxxx, perform more detailed diagnostics (and perform antibiotic susceptibility testing). nIt is necessary to follow the logical algoritm, for example like this: nGram staining à catalase test à plasmacoagulase test à STAPHYtest 16 etc. Exceptions: nASO is examined as other serological tasks (but important to know the meaning of the test for clinical practice) nG+ rods are not examined in this algoritmic way, but you get pictures and you have to say „this looks like Corynebacterium, this does not look like Corynebacterium, because it is spore forming“ etc. nVery similar is also a task to Clostridium tetani (to P07) Survey of diagnostics (simplified) http://www.ratsteachmicro.com/Staphylococci_Notes/HCOE_CAI_Review_Notes_Staphylococci.htm C:\Uživatel\Ondra\Obrázky a fotky\Odborné\Z internetu\Medici\Stafylokoky\29 Gram_Positive_Flow_Chart zmenšeno a inverze.gif Enterococcus or (or other tests) Corynebacteria, forms diftmik2 diftmik2a www.medmicro.info, Photo O. Z. raven wings palisade J07 Anaerobic jar description, explaining function C:\Uživatel\Ondra\Pracovní věci\Výuka\Fotoatlas\Anaerobní bakterie\Anae3.jpg air-proof lid palladium catalyser (beneath the lid) construction for placing of Petri dishes Anaerobiose generator (packet with chemicals) screw closer pressureventile www.medmicro.info, photo O. Z. C:\Uživatel\Ondra\Pracovní věci\Výuka\Fotoatlas\Anaerobní bakterie\Anae1.jpg www.medmicro.info, photo O. Z. source of anaerobic gases space for entering culture plates entrances for hands of personel Detection of lecitinase nLecitinase production is detected as strain precipitation on the yolk agar. Nevertheless, there are many lecitinases, and one only, that of Clostridium perfringens is interesting for us, we have to test, whether the lecitinase bay be inhibited by a specific antitoxin. „Negative I“ no lecitinase production. „Negative II“ a lecitinase is produced, but not the tested one C:\Uživatel\Ondra\Obrázky a fotky\Odborné\Kreslené\Lecitinase.GIF Detection of C. difficile toxin + – Test area Control area Survey of other tests, e. g. animal experiment nLook at the picture of tetanic mouse Opistotonus is typical both for mice and humans C:\Uživatel\Ondra\Obrázky a fotky\Odborné\Z internetu\Anaeroby\10 tetanus.jpg www.biotox.cz C:\Uživatel\Ondra\Obrázky a fotky\Odborné\Kreslené\Petrovy myši\mysh0001 bar.GIF Drawing by Petr Ondrovčík (1959–2007) Graphically adapted. Backgound counterstained using not malachit green, but „Paint“ programme by Microsoft Tetanic mouse P08: Ziehl-Neelsen stain: principle and knowledge about results (red bacteria on blue or green background) 32 Ziehl Neelsen www.spjc.edu 12 m_tub Ziehl-Neelsen stain www.primer.ru Another task: Culture of mycobacteria nHydroxide should be used before culture nWe use liquid Šula or Banić media and egg Ogawa or Löwenstein-Jenssen media. Egg media are solid because of egg white coagulation, they do not contain agar nResults are read after 1 (check for contamination) 3, 6 and for sure after 9 weeks of culture. (Positive results are mostly found after 6 weeks of culture.) n+ knowledge of more methods (PCR etc.) Appearance of mycobaterial colonies 21 mtbcolonies http://www.stockmedicalart.com/ P09: complete serology, plus knowledge of screening vs. confirmation tests Historical BWR – Bordet Wassermann Nontr. Screening RRR – Rapid Reagin Test or RPR or VDRL test MHA-TP (TPHA) Treponema Confirmatory ELISA FTA-ABS (indir. imunofluor.) Western Blotting Historical, or superconfirmation TPIT (Treponema Pallidum Imobilisation Test) = Nelson ouchterlony P10 – mycology: two tasks nOne of many ways, how to perform it, is microprecipitation in agar. It was already in J 07. Precipitation line is formed between the hole with antigen and the hole with antibody Holes with patient‘s sera 1–4 Hole with antigen positive Precipitation line – reason of positivity One is like P01–P06, another is this one P11: biofim Influence of saccharides presence to dental plaque formation: like in practical, but no 3D-graph is made nAssess the influence of uptake of various amounts of saccharides in food on rate of biofilm formation in a cariogenic Streptococcus mutans. nWhat are the conclusions of this experiment, as to amounts of saccharides in food, how long they stay in oral cavity etc.? Wells in the panel H:\Documents\Seropanl biofilm.bmp PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN Growth control Another task: MBEC assessment MBEC … minimal biofilm eradicating concentration Photo: Archive of Veronika Holá P12: Clinical microbiology I lFour tasks, all of them the same: l„For three minicasuistics, find suitable sampling methods and vessels/swabs for sampling“ lKnowledge of swabs and vessels necessary Some types of swabs F:\Documents\Klinika\11 Abstrichbesteck-MEDI_SWAB.jpg Amies medium with charcoal www.herenz.de Universal transport medium for bacteriology (all types of swabs). The wire variant important, if we want to go „behind the corner“ Plain (dry) swab www.calgarylabservices.com Today its use is for PCR and antigene detection only, not for culture! F:\Documents\Klinika\12 PlainSwab.gif More swabs F:\Documents\Klinika\17 virus swab.jpg Virus swab www.copanswabs.com F:\Documents\Klinika\18 chlamydia swab.jpg Chlamydia swab www.copanswabs.com Fungi Quick (for yeast and molds) www.copanswabs.com C. A. T. swab (for Candida And Trichomonas, from genitals only www.copanswabs.com P1010011čb P1010011čb Vessels Zkumavka sérová čb otočená bez popisu Sputovka čb otočená bez popisu Na střevní parazity čb otočený bez popisu Do toho se vychčije čb Common test tube. Universal use: clotted blood (serology), urine, CSF, pus, punctate etc.; blood and urinary cathethers, parts of tisue… Sputum vessel. Not only for sputum, but also larger parts of tissue etc. Stool vessel, for parasitology. Only this one does not have to be sterile! Vessel for urine sampling. It is better, if the patient urinates just into a test-tube, but especially for women it is difficult (except if they are in shower). So they can urinate into this vessel, and then a nurse removes the urine into a test-tube. P13: Clinical microbiology II nTwo tasks. One: Find a pathogen in oropharyngeal flora nNormal flora consists of greyish, viridating colonies (oral streptococci) and yellowish, usually non-haemolytical colonies (oral neisseriae). Possible pathogens are: nHaemolytic streptococci (and also Staphylococcus aureus) are visible by a strong haemolysis on blood agar nFor haemophili detection we use antibiotic disc with bacitracine – higher concentrations than in bacitracine test (to decline the normal microflora) – plus Staphylococcus line nFor meningococcal detection we use another disk, with mixture of vancomycin and colistin Task 1: Detection of pathogen in throat/sputum Očkování výtěrů 1 swab inoculation 2 loop inoculation 3 staphylococcus line 4 bacitracin disc (for hemophili) 5 V + K disc (colistine and vancomycine) for meningococci In all parts of inoculated area we search for colonies with haemolysis. They could be streptococci (rather colourless) or goldish) Cultivation result of throat swab with common flora Klin7 Klin9a In these sites we search for haemophili www.medmicro.info One more: Urine nTask: Perform semiquantitative and qualitative examination of urine nAs likely contamination (or accidental finding) is counted everything below 104 / ml, everything below 105 / ml in finding of two various bacteria and everything in three/more bacterial strains nAmong pathogens, the most common are enterobacteria, enterococci, S. agalactiae, staphylococci etc. Semiquantitative processing nA plastic loop is used – the „eye“ of the loop catches always 1 µl of urine nThis microliter is inoculated to one halfth of blood agar plate (you have it on a total plate) nFurther we inoculate Endo agar or URIchrom, here we assess it only qualitativelly nOf course, besides quantity examination we also examine genus and species of the bacterium as usually nIn our case, we would recommend nitrofurantoin for treatment. Semiquantitative urine evaluation Number of colonies Number of CFU (bacteria) in 1 µl of urine Number of CFU (bacteria) in 1 ml of urine Evaluation (valid for 1 bacterium) Less than 10 Less than 10 Less than 104 Contamination 10 – 100 10 – 100 104 - 105 Borderline More than 100 More than 100 More than 105 Infection See you at the examination! Klin2 www.medmicro.info