GENERAL ALGORITHM OF MICROBIOLOGY LABORATORY DIAGNOSIS The biological sample preheated from the infectious disease suspect case is investigated in ‘order to confirmiinfirm the infection and to identify the infection etiology. The etiology of the infection is identified using ‘A, MICROBIOLOGICAL DIAGNOSIS (direct, bacteriological diagnosis) B. IMUNOLOGICAL DIAGNOSIS (indirect diagnosis) C. MICROBIOLOGICAL DIAGNOSIS (direct, bacteriological diagnosis) ~ Its aiming to isolate and identity the etiological agent (the microorganisms which caused the infection) in the biological sample, as much as to test the antimicrobials susceptibility 1. Sampling and transport of samples: @. As soon as possible after the onset of the infectious disease, before starting the antimicrobial therapy (e.g. diarrhea - stool, meningeal syndrome - CSF etc.) 2. Direct examination of samples:: ‘a, Macroscopy ~ e.g. blue pus — Pseudomonas aeruginosa b. Microscopy — for orienting the following steps: i. 2-3 smears used for applying 2 staining methods: 1. Methylen blue — used for examining the cellular elements 2. Gram and 3. 1 smear to be kept for further examinations in case of ‘precious’ samples (CSF, punctions ete.) li, 1 smear used for applying Zeehl-Neelsen staining if mycobacterial infections are suspected (TB) THERE IS NO RECOMMENDATION TO PREPARE SMEARS FROM NORMALLY PLURIMICROBIAL SAMPLES ANDIOR FROM POSSIBLE PLURIMICROBIAL SAMPLES - e.g. FAECES (STOOL). FAECES — examined as wet preparation of the sample applied on a microscope slide covered by another thin slide — used for observing the presence of the white blood cells and/or of other elements which could providing information about the functioning of the intestinal tract. URINE - examined after centrifugation as wet preparation — for observing the presence of the white blood cells, of other normal or abnormal cells etc. FUNGAL INFECTIONS: wet preparation in 10 ~ 20% KOH-glycerol; India ink, nigrozine, May- Grunwald-Giemsa and Gram staining 3. Cultivation of the samples on microbiological media in order to obtain pure culture and isolate colonies a. Media and incubation conditions are chosen according the suspected etiological agent based on the clinical picture and the smear aspect b. Keeping correct aseptic techniques and correct methods of cultivation on solid media in order to obtain isolate colonies 4. Identification of the Genus and species of the microorganisms using pure culture and isolated colonies @. microscopy: smear presenting microorganisms with similar form and tinctorialty5, b. macrosoopy (the aspect of the culture): colony types: $ (smooth), R (rough), M (mucous), other characters (hemolysis, pigment etc.) ©. biochemistry: i. differential bacteriological media: contain the substrate and the indicator (or the indicator is added after the incubation) 4. antigenical characters using known antibody: e.g, slide ‘agglutination (Ag-Ac reaction; monovalent or polivalent sera) fe. pathogenicity: elaboration of different enzymes, pathogenicity on the experimental animals etc. (eg Bacillus anthracis, pneumococcus etc.) f. molecular biology methods other methods g. Identification of the subspecies (clone, strain) ~ typing methods - e.g a, Bacteriophage typing: bacteriophage definition = virus infecting bacteria based on the presence of bacteriophage receptors on bacteria cel wall b. Genotyping (molecular typing) 6. Antimicrobial susceptibility testing for bacteria andlor fungi — usually disc diffusion method, sometimes MIC (Minimum Inhibitory Concentration) and other special techniques for investigating the ‘in vivo" efficacy of antimicrobials ‘A. IMUNOLOGICAL DIAGNOSIS (indirect diagnosis) B1. serological diagnosis ~ using known microbial antigens (Ag) we are looking to find specifio antibodies (Ac) produced by the organisms against the suspecied etiological agent We The presence of the Ac Their dinamic (innitialy and after 7-10 days) The type of the Ac - M class Immunoglobulin, at the beginning of the disease, then G class immunoglobulin in case of thymus-dependent antigens B2. The study of the patient's reactivity to different antigens by intradermal reaction B21. In vivo Candidine i.d.r. ete. 82.2. In vitro tests for cellular type immunitySAMPLII General ING OF BIOLOGICAL PRODUCTS AND TRANSPORT OF SAMPLES rules: 1. Under the supervision of a specialist 2. Before starting the antimicrobial therapy or: ‘a. stopping the therapy for 24h, if possible . sampling immediately before administering the following antimicrobial dose ©. adding antimicrobial antagonists in the bacteriolagical medium: e.g. Mg sulphate for Tetraciclines, Penicilinase, resins, activated charcoal eto inoculum dilution 3. As soon as possible after the onset, but in the most probable period for the isolation (€. 9. during the shivering — bloodculture (BC); after the frst week from the onset in the typhoid fever — need to know the evolution of every infectious disease) 4. The sample has to be chosen according to the clinical manifestations and the physiopathology ofthe disease: a. From the place were the microorganism is entering the human body (entrance portal) b. From the ways of propagation into the organism . From the ways of elimination out the organism (exit portal) d. From the infectious process 5. Indicated periodicity of sampling a. bloodstream infection (2-3 samplings in 24 h) b. stool (3 consecutive days) . lung TB (3 consecutive days etc.) The sample must be the one indicated in the sample report form The most representative samples have to be used (ex. pus, blood, mucous parts ofthe stool etc.) 8, The quantity ofthe sample has to be sufficient 9. Aseptic technique and precaution to avoid : @. Contamination of staf involved in sampling biological products b. Contamination ofthe sample ©. Suprainfection ofthe patient d. Contamination of the medium Ao LABORATORY MATERIALS FOR SAMPLING: New and Sterile bottles or other recipients, free of inhibitors, safe locked Appropriate materials (ex. do not sample with cotton for Bordetella; itis inhibitor) Laboratory recipients have to be marked ded information to appear in the clinical register, sample report form, laboratory register laboratory report form: Patient identification data ‘Sample identification data — unique identification number Date and place of sampling, who was sampling Type of sample ‘Suspected clinical diagnosis, date of onset, medication Information about the condition o the transport Date and hour of the reception in the laboratoryThe sample will be refused in case of = Missing data; some data could be recovered by phone = Poor qualty of the product, if there is a possibilty to repeat sampling ~ Bronchopulmonary secretion from 24 h (pool) ~ Saliva instead of inferior respiratory tract secretion + Urine, ifitis sampled more than 1 h before ~ Exudates, inferior respiratory tract secretion, faeces, if there is a requirement for anaerobic microorganisms ~ Clearly contaminated samples (poor condition ofthe recipient) TRANSPORT AS SOON AS POSSIBLE (Mx 1-2 h FOR THE MAJORITY OF THE PRODUCTS) Keeping the adequate temperature (e.9. 37 Celsius degrees for Neisseria meningitidis) Free of transport medium, if possible, if not transported at big distances Avoiding the contamination Sometimes sampling and cultivating near the patient bed (e.g. Blood Culture) If the fast transport is not possible — preservation: Isoterm container 0-4 ° C - NOT FOR Neiseria, Haemophilus ~ Transport medium ~ Antimicrobial supplement for fungi isolation: peniciline, streptomycin, cloramphenicol = _ Into the syringe, eliminating the air, for isolating anaerobes Advised courrier INTERNATIONAL RULES FOR PACKAGES —triole package rule ~see official marking on net Examples of samples: NB — blocdculture, urina culture and stool culture will be discussed separately, during the second semestreBunes fq patdwes souesqwew ‘eros uy Jejnouio Aq auop siBuydweg uado epi wnow ay Jossaxdap ‘enBuo} :sjvauunyjsuy Buoddng - (yBnoo) Buydwes Buunp UOeUINE}UCO ialyiyd 1408 '3NONOL 104 "eps WOU :LON ‘SiIS Gems yequu} ‘SIISUO} uoridsns | 94) Buydwes ‘sued xutseud Buyeo euatyudip jo aseo * | sousised auy uo | saye y p seat wy 9 U€ ‘uoge|noout ‘9 Suoyesa9)n | swisueBve6 ynowM eIpaU Jo} ‘sysodap snd uo 1YSPM Ya} ueL xn} —2 ‘Adcosoiw Uwugy Yn'seucz | nowy ‘Bupyuup Age paul | yeup soy :sgems res wae © Powweyu | 40 Buyes a1oeq uinipaw satuy ‘aIqRsayalg uoyoo (€) Z - (UOYISOq - | WON ajepnsx3, e gems Oy | wnjpeur wnipaw | yodsues jnouym ‘soajnep wodsues, | ‘yodsues jo ow, Buyduieg MoH uM oun od aides® ‘ain o voyeumnya ‘ayetduico Yun “segoieeue 0, abuuise Susy « ~ (sasseage deep) uonendse w Jo voyound +-So'O:Aguenp ‘abejauno SISK “fecoa panes 830 Aq ‘anedid uayis ewipAsana “qe suowBajyd soqavceue uzsam| svaseaiodoy — | gens fa Buxdues “sassgoge 20} SJ2UIE|UCO |eKDads- ‘AeyeIpeuiu) [eo\Go}0u8}eq Plone- uojezi}290) ‘sung ‘spunom 2 uiypow samy one wm uoBupued + | paca woy sng | wesaid ssn vou, sna ‘Sumids Aq padwes sevesquaw "esos ‘uy sejnauio Aq. oucp s 6urdues + ado pt 4204 0) weed + vooidns “amnoedoid evowgudipo 9c u sosseldep E ‘onfiuoy :siuawnaisu, "oj 2 ‘boo. 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Accomplished usually by: heat, filtration, but also by chemicals and radiations Disinfection = destruction, inhibition or removal of microorganisms which may cause disease or another problems. Accomplished usually by disinfectants. Asepsis = procedures which avoid contamination with infectious agents of the surfaces, culture media, skin, mucous membranes etc. Antisepsis = application of antimicrobial substances with non-selective antimicrobial effect, called antiseptics ‘Antiseptics may by applied on skin and mucosa, disinfectants only on inorganic materials. HEAT STERILISATION ~ DRY HEAT: heating of the loop in the flame, the Poupinel or the Pasteur oven - WET HEAT: the autoclave, tyndalization; incomplete sterilization - pasteurization — principle, types of pasteurization Using the autoclave Principle: use of pressure steam to produce a temperature of 121°C, maintained for 15 minutes, in order to kil all microorganisms, including the bacterial spores. Sterilization at 0,5 atmospheres (115 °C, 30' to avoid carbonizing of sugars; sterlizations for very dirty material at 134 °C (2 atmospheres) Sterilization control: by physical, chemical and biological RADIATION STERILIZATION: UV LAMP (nucleic acids damage: functic = lonic radiations jing interval is limited in time); FILTERING STERILIZATION: ~ Filter membranes + Different other porous materials ~ Biosafety cabinet filters (HEPA = High Efficiency Particulate Air Filters) Disinfectants Important choosing, preparing and using disinfectants Its recommended to use disinfectants specific to the scope at specific work concentrations, ‘Spaulding pyramid (see the course) Usual disinfectants and their use Disinfectant [Use Concentration Virkon Work surfaces, containers for | 1% viv pipettes or slides, skin | Powder disinfection, spillage Hypochlorite (sodium chlorate |) [conttiners for pipettes and | 2500 ppm (0.25%, viv) slides Free chlorine Ethanol Skin antisepsy 70% viv industrial methylated | alcohol Protection glasses and gloves during the preparation ofthe solutions 1Freshly prepared solutions (from stock concentrated solutions or powder) Virkon ~ 1 week availabilty - preserving pink color means i stil effective; Bleach (hypochlorite) -a fresh solution must be prepared every day Other disinfectants: phenols, cationic detergents DECONTAMINATION and FLOW OF MATERIALS IN THE MICROBIOLOGY LABORATORY STERILIZATION OF LABORATORY EQUIPMENTS AND MATERIALS The loop Heating to red inthe flame, the Bunsen ges lamp or other heat source Glass bottles, glass pipettes, glass Petri dishes ~ Pasteur oven, covered with special paper, free of grease, or with aluminium folle - 2h at 160°C, Autoclave: ‘See course lecture on stelisation Microbiological culture media and solutions, plastics resistant to autoclave sterilization, cotton ete. Autoclave - see course lecture on stelisation Glass sticks and metal scissors ‘Avcohol burning (industrial methyic alcohol 70%) Laboratory dangerous waste must be automatically broken in pieces and autoclaved: infectious materials, biological waste, laboratory animals etc, In Romania it is mandatory to use level 4 decontamination treatment (i.e. - the number of microorganisms must decrease by 6 logs 10.