Republie of the Philippin @ ) Pina ca oalbalciil Ades NL Ree a uk uNIvt raced BACTERIOLOGY A LABORATORY MANUAL Instructor/ Author College of Health Sciences— een INSTRUMENTATION IN MICROBIOLOGY LEARNING OBJECTIVES Once you have completed the experiments in this section, you should be 4, Familiar with the tools and equipment used in Microbiology Laboratory. nits, use, and care of each instrument. 2 Able to understand the componer safety ina 3. Able to know and understand the organization and general s clinical microbiology laboratory Introduction Microbiology focuses on microorganisms, including bacteria, fungi, protozoa and viruses, and their effects on other living organisms. Studying these effects is helpful for developing new medications and cures for illnesses. Diagnostic medical mierobiology is eoncered with the etiologic diagnosis of infection Laboratory procedures used inthe diagnosis of infectious disease in humans include the following: |. Morphologic identification ofthe agent in stains of specimens or sections of tissues. 2 Culture isolation and identification of the agent. 3, Deiection of antigen from the agent by immunologic assay latex agglutination, enzyme immunoassay by ‘luorescein-labelled (or peroxidase-labelled) antibody stains 4. DNA-DNA or DNA-RNA hybridization to detect pathogen specific genes in patients’ specimens. '5, Detection and amplification of organism nucleic acid in patients’ specimens. 6 Demonstration of meaningful antibody or cell-mediated immune responses o an infectious agent In the field of infectious diseases, laboratory test results depend largely on the quality of the specimen, the timing and the care in which itis collected, and the technical proficiency and experience of laboratory personnel, Although. physicians should be competent to perform a few simple, erucial microbiologic tests—make and stain a smear, Examine it microscopically, and streak a culture plate—technical details ofthe more involved procedures are ‘usually left tothe bacteriologist or virologist andthe technicians on the staf. Physicians who deal with infectious processes, must know when to and how to take specimens, what laboratory ‘examinations to request, and how to interpret the laboratory results. In order to run their tests, scientists need ‘samples of microorganisms, and they need containers in which to hold and study them. The tools used in a ‘microbiology lab help scientists carry out their tests and analyze their result.Sterile Specimen Bottle Sterile tongue depressors andl swabs Microscope Gilass Slides Applicator sticks Patti Dishes Tnocutating Loop Inoeutating, Neale Runsen Burner + Pasteur pipettes st Tubes Serew Cap Test Tubes AW LAND DISCUSS ITS FUNCTION Autoclave Clinical centrifuge Incubator Fume Hood Refrigerator Candle Jar Antibiotic Disks Blood Culture bottles Microscope ia Mlustrate and label accurately, and give the funetion/s of each of the different materials and equipment listed above which are used in Microbiology laboratory, 1. What are the Routes of Infection in the Microbiology Laboratory? Frmamerate the General Considerations for Laboratory Safety that mast be followed in any linc laboratory, 3. What are the biosafety categories classified by the CDC ~Y How are they categorized?COAT : Se FUNCTIONS | Provides protection oF the skin and personal clothing during the handling of chemical hazards from incidental conjoct and smallsplaches- Also, it : rovides a detachable bamer In the event of an accident involving Spill or splash of hazardous cubstancts y explosion and Other unexpec- ted ewenis thot ean possi ly occur inside the laboratory: Masks help to prevent spread of dropleis by the Person wearing them. they also prevent sploghes or Sprays from reaching the person wearing the masks making them useful for cleaning up hazardous Substance spills.BACTERIOLOGY C Ge stent Tongue Oepressors and Swabs Microscope Glass Slides FUNCTIONS iis intended for the colle ction of biological specimens such os es, sputum, periton eegotesjoint aba and biopsy specimens for JaboratOry analysis- Sterile Tongue Depressors may de used to Facil tote insertion of the ov airway by preventing folding oe the tongus which may impair venous and lymphatic drainage and results $0 dirway obstruction. Sterile wad precerveeniie integrity of the samples durifg handling and trans- Poriation to int laboratory: A glacs slide is @ thin, flat rectangular pitce of ae that is used asa platform for microscopic specimen observation. Usuall measures 25 mm Ebi) TOmm or 4 inch Dy F inches lon i i to fil undead Hoqemaa Clips of @ microscope.BACTERIOLOGY A LABORATORY MANUAL Mustrations FUNCTIONS Applicator Sticks : Applicator Sticks are used to mix body fluids with a reagent or to prepare slides for Micro scopy. DISHES A petri dish is a shallow cylindrical, round glass uied in labs to culbure Varjous bacteria and cells. 1 is necessary to keep microorganigms likt bacteria and viruses separated from other species or elements whilt examining them petRI An inoculating loop is a Simple toot uted mainly to pick UP and transfer a small famplt from o Culture of micro organisms, bre Sa eR Desai ~— are & 0 transte TNOCULATING NEED, solid media to. other waite media ov pedi plates. Commonly used for retrieving soli materi ny a or dense ct pOBACTERION ~ LsoRaTont neta. Fun trons Bunsen burner is a source of open flame that is usd Barrel to sterilize loops and needies ,as well as flaming tht tips of jest hic Intgke tubes during Inoculation. Holes Mustrations BUNSEN BURNER Gas Valve Collar Needle Value Bast ° Also Known as tye dfopper PasTEUR is ag devict used to irangfer PIPETTES small quantities of Nquids- mo are Used in the labos ratory and also to dispenct smallamounts of liquid medicines. Tes} Tube iv a glass TESt TUBES container used in a scinn- tiffe laboratory. Also used for heating 4nd holdin g Chemicals during exp crime nts Sorew cap test tubes are made up of borosilicate glass and cyitable for transporting Gnd growing of culures- SCREW CAP TES1 TURESBACTERIOLOG A LABORATORY MANUAL Mustrations FORCEPS SYRINGE i y PLUNGER R N Con MAL SALT ap NE Qa i} FUNCTIONS Forceps are used when Fingers are 100 large 3. Grasp small objects o When many objecic needed to be held at one time in a laboratory Syringe are offen used for measuring and transferring solvent S and reagents where a high prcsioy is not required: Normal Saline is ured for the preparation of culture Suspensions or Serial dilution in microbiology. Culure suspensions are pre Pared in Saline polution because 0-9%6 sodium chloride sowdion ic isotonic in nadure. Wash Botte Is a squeeze bottle with a nor2Ie, used to rinst Various Pieces of Labarnen Glassware cuch as tert tubes and found bottom flasks. ELBACTERIOLOGY ORATORY MANUAL Mustrations cover Rody Jacket 11 Bucket Stand Ors PLi PCB IMBALANCE CENTRIFUGE PEYECTION mMoTOR AssEMBly Safety Valve Rerease Valve Cover tig thening Pressure Gaugt They are used to FUNCTIONS hutoclaves operate at nigh temperature and pressure fo kill micro - Organisms and cpaiees econ- taminate certain bio~ logical waste ana Steri- lize media, instruments Ond labware. ab orad qn emic, clinjeal to hn and ured to » fu cellular rganelies » viruses, rottins and nucleic acidsTENPEROTURE INDICATOR Bigh/Low FAN \ inrernae partic pIR LOCK DOOR SUCTION OPENING FUME HOOD __ | | | 11 chpinet — @oov ACCESS DooR al Til | CONTROLLER hy T TOBLY Tor ¥ 2 ¢ SINK @ND TOP a sl = = FOUR apiusTiNG PP ANTIe HINGE’, BLUB Petr QUERFLOW PP MATERIAL STRIP Ons POWER INDICATOR HEATING Juicy DUMIditYy and FUNCTIONS | an incubator is htated , insulated box Used 40 grow And maintain microbio- logical or cell cultures. the intubdtor maintains optimal temptrajure, gOseous fontens of the atmosphere inside. Fume hoods are ventilated enciosures thad temove hazardous chemical fumes 290 Volatie vapors trom the labora tor Toviding Personner pro teey ou .sueFUN CTIONS oe Digital 1B SE Refrigerators, are : Le DISPLAY decigntd to store comples, POWER WLI Specimens ,Vaccines an Ginss DOOR Medicines ata very Speake tonpt Ta range they are used to cool samples OF specimens for preservation. sf Drawer caster Candie Tar ore used 40 grow bacteria requiring on increased COa, concentration . It increases COa concentrations and stillleaue Some O. for Qerobic Caprophiies. CANDLE GAR Antibiotic disks are used to Getect the cusceptibilit y OF @ @ bacterial iro lates to antibiot @ ANTIBIOTIC DISKBACTERIOLOGY ‘A LABORATORY MANUAL Mlustrations Bloo E BOTTLES FUNCTIONS ee Blood Culture vO contain a grow medium Which helps. in the growth of hacteria, ac well Os an anticoaguiant which inhibits blood clotting. . Microscopes are used to MICROSCOPE a Vaentitycand visualize come —— EYE PIECE samples including bacteria, Algat and Fung): Generally, it ls Used to observe small ROUGH ADTUSTMD jects shad are not ceenby Nose PIECE the Naked tye. obJtctive LENtES cur —— JTAOE Body TUBE mor Clinical Bacteriology LaboratoryBACTERIOLOG ‘A LABORATORY MANUAL Naas ‘COURSE /YEAR/SECTION SCORE, pepiamaa Redan FO LOND ES AOR (Ce lO eee INSTRUMENTATION ON MICROBIOLOGY Question for Research , P ; t ') What are the routes of infection in the microbiclo gy laboratory! “Inhalation (atrosols) + Pereutantous Inoculation (needit and fyringt cuts o¢ Abrasions from contaminated items and animal bites ) “Contact between mucous membranes and contaminated material(hands or surfaces * Ingestion acpiration through a pipettt.emoking or eating) REFERENCE: Laboratory acquired infections and bio- incidents (aoa). Belgian eiesatkty Server, Retrieved from ntt0s+// www. bi osarety- be / Content /1a boratory - AC Quired-infections -and-bid- inci dents Enumerate the General Considerations for laboratory satety that musi be followed in any clinical laboratory: + Do not conduct the ‘what if” question + Accume that substances of unknown toxicity are hazardous, * Be alert to unsafe conditions and ensure that they are Corrected OF soon ar they are detected. +Make cure that all Containers are bepenel loboeied: +a fume hood of other containment devict must be used if typosure limits are likely to be ey ceeded: + Avoid *sniff testing” chemicals + Wear appropriate Personal Protective E + No Food, Tobacco and Cosmetics + Avoid self: injection + Never taste chemicals + Never Ule mouth suction to pipette chemicals * Only Wie glassWare or utensils for their inte 3 . Hondle aed store laboratory glassware Dinieead Canes AVOIO the behavior of horetplaying Ond behavior that is distuptive , agressive, or in any Way presents @ hazard to those working in the laboratory. REFERENCE : General Lab safety Rules (n-d) Chemical Hy glen oe Retrieved tom nttpss//wp. stotaf-edu|chemical- hygiene /fatetu-ruter/ Eee eee Procedure unless you ean answer all s. Wipment at ai times.‘Question for Research 8) What are blocatety categories classified by the COC? How Ore they CAteGorizedt Biosatety Level 1(BSL-1) *BSL-1 Jabs ore used to study infectious agents ov toxins not known to consistently couse disease in healthy adults. they follow basic safety procedures called Standard Microbiological Practices and require no special equipment of design features: Standard Engineering contrals in BEL-1 laboratories include €asily Cleaned surfaces that are able to withetand the basic chemicals used in ine laboratory. Biosafety Level 2( 8st -a) They are used to study moderate -risks infectious agenis or toxins that posed a risk if accidentally inhaled , swallowed or eyposed to the skin. Design requirements for Bst -4 laboratories include hand washing, eye washing siations incase of accidents, and doors that close automatically Gnd lock. BSL-2 labs must also have access to equipment that can decontaminaie laboratory waste including an incinerator, autoclave and/or another mtthod ,depending On the biological risk acsetcment. Biosafety Level 3 (RSL -3) BSL-3 laboratories are used to stud Yoxings dat may be transmiited to air and can Cause Potentially lethal infection through inhalation evpocure- Researchers perform all experimenis in biosately cabinels that Use Carefully COniroiled air flow or sealed enclosures to prevent infection: they are designed to be easily decontaminated - these laovoratories must use controlled, or direCtional, air flow to ensure thot air flows from non laboratory areas (fuch Os the hallway) inio laboratory Oreos as an additional safety measures. other engineered safety features include the ure of two self-closing or interlocked doors sealed Windows and wall surfaces and Filtered ventitaiion systems. Biosotery Level 4 ( BSL-4) BSL-4 laboratories are used to study infectious agents Or fOxiNS that pose a high risk of agrosol-transmithed laboratory infections and life-threatening diseas which no vaceine / therapy is availanle, the Lab a Incorporate gil BSL -3 features and OCCUPY cate Peolabedk Zones within o larger building or may be housed in ® Separate, dedicated building» Access to Sst -4 eee y infectious agents orBACTERIOLOGY A LABORATORY MANUAL Jaboratories is carefully controled and requires significant training + There are two types of BSL -H laboratories + “Cadinet Laboratory - all Work with infectious agents / toxin’ is done in a Class Il] Biosafety tabinet with very Carefully epg nes Procedures to contain ony Potential Contamination. In addition 10 being designed to also prevent Contamination Of other spaces. ‘Suit Laboratory = requires all laboratory personne! ore required Yo wear full -bady, Air supplied units, which are the most Sophisticaied type of perronal protective tguipment. All personnel showey before tviting the laboratory and go through o series Of procedures designed procedures 40 sully Atcontamin ate hem before leaving. The pnanetting controls required are different for BSL -4 Cabine} and suit laboratories. For either type, they art tutensive and supplemented oy carefully olesigne procedures and practices REFERENCE: Biosafety Levels (n-d) Science Safty Security. Retrieved trom https: www phe gov /#3/ BioviskManagement fo Biosafety -Levels . aspx iocatety/Pages/ mon Clinical Bacteriology Labo