TRACING THE CRIMINAL Part fourteen: Repeating Institute for microbiology shows L Organisation of practical exam •Usually student picks one of 50–60 tasks •One 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 neutralization and streptococci • Some tasks are mostly practical (like Gram staining), some are rather discussion with practical parts. Students may be asked also to topics not directly placed in the tasks J01 Microscopy •Three tasks •Wet mount – for large and/or motile microbes (parasites, fungi, motile bacteria) –To know also: Dark field wet mount (mainly spirochets) •Gram staining – how to do it, + survey of other staining methods (Giemsa, Ziehl Neelsen, Burri…) •Observation of already stained preparations – mainly interpretation Wet mount – do it practically •Do not forget to cover the preparation by a coverslip and to use non immersion objectives, magnifying e. g. 4×, 10× or 40×. •We use no immersion oil •After having it done, observe the objects in the microscope •Attention! Students that put into contact a non-immersion objective and immersion oil, finish immediatelly with F!!! 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 •Gentian/crystaline violet = Sol. Gram-Nowy (20 –) 30 sec. •Lugol (20 –) 30 sec. •Alkohol 15 (– 20) sec. •rinse by tap water!!! imporant! •Safranin 60 – 120 sec. •rinse by tap water •dry by filtration paper •microscopy as in Task One Dift1 P1010003 Specimen microscopy Strain microscopy Photo O. Z. In observation of slides… •…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!) •For example: –WBCs = inflamation –no WBCs in sputum = not properly taken specimen –G- diplococci inside WBC, urethral swab – suspicion for gonorrhoea J02 – 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 •1. Broth •2. VL-broth •3. selenite broth •4. Sabouraud •5. Löwenstein-Jenssen •6. Blood agar •7. Endo agar •8. MH •9. 10 % NaCl •10. VLA •11. XLD (+ MAL) •12. CHA •13. Levinthal •14. Slanetz-Bartley Some more media at special bacteriology tasks. J03 – biochemical identification tests One only task, but other stuff is in other topics! •In special bacteriology, you might meet: –Catalase test –Tests with diagnostic strips (oxidase, PYR, INAC) –Hajna medium (red = G-NF, other = ENT/VIB) –Eventually also MIU (not necessary to know details) •The only „pure biochemical“ task –ENTEROtest 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 – J04 •Important basic information: •If 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. –In testing of disinfection effect, bacteria are treated by a disinfectant and then cultured on a medium without disinfectant –In 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): •An extra sub-task to both tasks: Make pairs of cards with names of methods/disinfectants and cards of characterisation of methods •The cards are on a working table, it is possible to see it J05: Atb susceptibility Diffusion disc test: to read it, to interprete it atbpsae21 www.medmicro.info Microdilution test – reading •In case of collumns 1, 3, 4 & 5, MIC > the highest value •In 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 •We 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. J06–J08 (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 •Antigen detection (including antigen analysis): it is a direct method. Positive result means presence of the microbe in the pacient‘s body •Antibody detection: it is an indirect method. Nevertheless, there are some ways how to get the information – when the microbe met the body: –Amount of antibodies (relative – titre + titre dynamics; agglutination, CFT, neutralisation) –Class of antibodies: IgM/IgG (reactions with labelled components – mostly ELISA and immunoblotting) –(Avidity of antibodies) Precipitation Precipitace Two examples of precipitation •1) Microprecipitation in agar according to Ouchterlony ouchterlony + - - - 2) RRR/RPR reaction for syphilis diagnostics (flocculation) Agglutination Aglutinace Agglutination for antigen detection or antigen analysis •EPEC 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) •CSF 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 •K+ positive, titre = 1 : 200 •No 1 negative •No 2 posit., titre = 1 : 400 •No 3 negative •No 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 •Antibody (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 •After every streptococcal infection antibodies are produced, often including antibodies against streptococcal toxin – streptolysin O. •Nevertheless, 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 •In such a situation, paradoxically the antibodies are worse than the pathogen that challenged the antibody response to protect us. HIT •Haemagglutination Inhibition Test: Pay attention, it is NOT an agglutination reaction, it is a neutralisation! Antibody neutralises the aggregation of RBCs due to viruses. •So: Potato-like shape = negative response. Dense round target = positive response •HIT 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 •HBsAg testing – positive •HBsAg testing – negative •anti-HBs testing – positive •anti-HBs testing – negative • 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 •1: original antigen (mixed) •2: decomposition of antigen by a detergent •3: electroforetic division of antigen •4: „blotting“ of divided antigen to a nitrocelulose membrane •5: 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! •It is necessary to know not only how to read the test, but also how to interprete it. •There are two special tasks (concerning toxoplasmosis and Lyme disease) based on complex interpretation of all results including anamnesis! –E. g. pregnant woman with IgG anti-toxoplasmosis is NOT ill, but protected! J09: DNA detection (PCR) Mostly use of PCR in medical microbiology •The methods are used mostly in situations, where microscopic and culture diagnostic is difficult or impossible •It is not very useful for common, ubiquitous pathogens. Because of its sensitivity they would detect accidental molecules comming from environment •The 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 J10+11: viruses •Majority of viral tasks are serological examinations (HBsAg, anti-HBs) •Two extra virological tasks concern isolation of viruses –Fertilized egg – parts of fertilised egg, used for isolation –Cytopathic 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 J12 Parasitology •As a basis, we use methods based on modified wet mount: –In Kato method counterstain with malachite green is used, to make parasites better visible –Faust method is a concentration one (see later) •Graham method is used in pinworms only (and as one task you can do it practically!) •Wet 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 •You 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 •P: healthy pregnant woman, cats at home •Q: another healthy pregnant woman, no cats •R: a young lady trekking in forest; no cats, but contact with objects comntaminated by faeces of wild animals •S: a senior, working in garden, cats use to walk throught the garden, symptomas of retinitis + enlarged lymphonodes ouchterlony J13 – mycology: two tasks •One of many ways, how to perform it, is microprecipitation in agar. It was already in J06. 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 (see later), another is this one J14: biofim Influence of saccharides presence to dental plaque formation: like in practical, inculding 3D-graph making •Assess the influence of uptake of various amounts of saccharides in food on rate of biofilm formation in a cariogenic Streptococcus mutans. •What 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 P01 to P06, J13 •There is a universal task to those topics: •„Among given strains, find strain(s) of Xxxxx, perform more detailed diagnostics (and perform antibiotic susceptibility testing). •It is necessary to follow the logical algoritm, for example like this: •Gram staining à catalase test à plasmacoagulase test à STAPHYtest 16 etc. Exceptions: •ASO is examined as other serological tasks (but important to know the meaning of the test for clinical practice) •G+ 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. •Very 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 •Look 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 •Hydroxide should be used before culture •We 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 •Results 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.) •+ 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 P10–P13: Clinical microbiology I-IV lSeveral 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. Clinical microbiology (continuing) •Several more tasks. One: Find a pathogen in oropharyngeal flora •Normal flora consists of greyish, viridating colonies (oral streptococci) and yellowish, usually non-haemolytical colonies (oral neisseriae). Possible pathogens are: –Haemolytic streptococci (and also Staphylococcus aureus) are visible by a strong haemolysis on blood agar –For haemophili detection we use antibiotic disc with bacitracine – higher concentrations than in bacitracine test (to decline the normal microflora) – plus Staphylococcus line –For meningococcal detection we use another disk, with mixture of vancomycin and colistin 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 •Task: Perform semiquantitative and qualitative examination of urine •As 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 •Among pathogens, the most common are enterobacteria, enterococci, S. agalactiae, staphylococci etc. Semiquantitative processing •A plastic loop is used – the „eye“ of the loop catches always 1 µl of urine •This microliter is inoculated to one halfth of blood agar plate (you have it on a total plate) •Further we inoculate Endo agar or URIchrom, here we assess it only qualitativelly •Of course, besides quantity examination we also examine genus and species of the bacterium as usually •In 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 Some more tasks •Examination of stool (observation of result on various media) •Examination of vaginal smear, Nugent score counting •Examination of vaginal swab (culture) •Examination of wound swab (sampling, moulage-imprint method) •Examination of blood cultures (microscopy + culture) See you at the examination! Klin2 www.medmicro.info