Lab 2 lab 3


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Laboratory safety for 3rd year medicine students for Clinical Microbiology. Microscopy and Bacterial Morphology

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Lab 2 lab 3

  1. 1. Microbiology practical 3nd year medicine Clinical Microbiology 1st Principles of BacteriologyOBJECTIVES 1. To know and understand the principle of bacteriological identification tests . 2. To know what specimen to take from a patient and what test (s) to request from the bacteriology laboratory. 3. To be able to interpret the results of laboratory tests to aid in the final diagnosis. UQU Micro. Raniah 1429-1430 1
  2. 2. BASIC L LABORAT TORY SA AFETY M MEASUR RES You m be stric adhere to it, an so you can protect yourself, your colle must ctly ed nd c t eagues and d your famiily. FOLL LOWING SSAFETY M MEASURE CONSI ES IDERED I YOU LAB EVAL IN LUTION. 1. We a full length zip ear pped up w white lab coat. 2. We gloves when working. ear 3. No open sho allowe o oes ed. 4. Lo hair must be tied back and da ong k, angling je ewelry an baggy nd y clo othing mu be secu ust ured. 5. No eating, s o smoking o drinkin in the lab. or ng 6. Do not put a o mouth e.g. pencils, p anything in your m pipettes, f fingers, lip balm. p 7. No personal belongin allowe to the lab (e.g.; bags). o l ngs ed l 8. Be efore leavi the la remov your co hang it or put it in a separ bag. D ing ab, ve oat t rate Do not wear you work la coat out t ur ab tside the departmen d nt. 9. Keeep your area o work tidy put away ite you are finish r of t ems hed wi ith, dis scard wast material you have finished with. te l e 10. De econtaminate your area with the prov h vided disi infectant ( (Dettol co oloroxylen nol 50% 20%), or hypoch %- hlorite. 11. Sm spilla must b moppe up with disinfect mall age be ed h tant. efore and after the lab time). 12. Wa your hands (be ash d eACCIDE ENTS AN INJUR ND RIES: 13. Report any a accident (sp breakage, etc.) or injury (c burn, etc.) to the superviso pill, o cut, e or imm mediately.. 14. Tre minor c and ab eat cuts brasions a follows: as a) Wa thorou ash ughly. b) Alllow to ble freely. eed c) Dr and apply elastopl ry last. d) En details in acciden book an notify th lab seni nter s nt nd he ior. 15. If y or you lab partn is hurt, immediat you ur ner , tely yell o the sup out pervisors. Do not pannic. 16. . If a chemical should splash in y f your eye(s or on yo skin, im s) our mmediatel flush wi ly ith runnning wat for at l ter least 20 m minutes. UQU Mi icro. Raniah 1429-1430 h 0 2
  3. 3. 1.INTRODUCTION TO LABORATORY SAFETY1. OBJECTIVES:• To get familiar with general safety measures considered necessary to work I n a microbiology laboratory, and when dealing with basic medical (biohazard) waste.• To make awareness of how important is the use of safety cabinet in the handling of infectious material, in particular microorganism.2. BACKGROUNDa wide variety of specimen are received daily in a microbiology laboratory, many ofthem containing pathogenic microorganisms. Laboratory acquired infections havealways been a hazard for those working in the medical laboratories and similarenvironment. Infection in microbiology laboratories is generally caused byinhalation, inoculation, or ingestion of microorganisms. Release ofmicroorganisms in the form of aerosols increase the risk of infection by inhalationunless the aerosols are contained or restricted. Aerosols can be created; by culturingmicroorganisms, particularly from fluid specimens; by accidents, such as breakagein a centrifuge; or by the dropping of culture. To contain aerosols, “safetycabinet” should be used for all manipulations likely to produceinfected aerosols.ROUTS OF ENTRY: (HOW WE GET INFECTED)Infection in microbiology laboratories is generally caused by1. Inhalation (through the lung) inhalation of airborne microorganisms 2. Inoculation (conjunctiva, skin injuries) skin - injuries by needles, sharp instruments, or glass. Animal bites and scratches. Cuts and scratches. conjunctiva - splashes of infectious material into the eye, transfer of microorgansims to eyes by contaminated fingers3. Ingestion (mouth) through eating, drinking, and smoking in the laboratory, mouth pipetting, transfer of microorganisms to mouth by contaminated fingers or articles. HAND WASHING Hand washing in right way and right time is basic andimportant procedure in protecting the laboratory worker as well as the patient andthe hole community form getting infected with so many dangerous infectiousdiseases. Especially those microbes which can be easily transmitted via directcontact. Figure (1).Safety is the responsibility of every member of the laboratory including the head ofthe department. Although safety on laboratories relies predominantly on the commonsense of individuals, it is necessary to lay down general guidelines and rules, whichmust be in practice at all times. One such guidelines is prepared and attached hereUQU Micro. Raniah 1429-1430 3
  4. 4. for all the students and supervisors to follow while they are working in the laboratory(page 2 ).Clean HandsSave Lives. Note: Rubbing with soap (step 1 t0 6) should be done for 20 seconds. And repeated if needed tell the hand is clean Figure ( 1 ): Hand Washing.UQU Micro. Raniah 1429-1430 4
  5. 5. 2. MEDICAL WASTES BLACK Or TRANSPARENT BAGS • PAPERS , WATER BOTTLES…etc (basket OUTSIDE THE LAB)A. CONTAMINATED MATERIALS • GLOVES Yellow Bags with biohazard sign. • CONTAMINATED PLASTIC LOOPS Discard Jars with disinfectant. • CONTAMINATED WIRE LOOP Red hot (flaming) • CULTURE PLATES WITH GROWTH Yellow or Red square basket. • INFECTED SHARPS (Syringes Needles, Glass Slides) Sharp container clearly labeled (NSI).UQU Micro. Raniah 1429-1430 5
  6. 6. 3. BIOLOGICAL SAFETY CABINETS The biological safety cabinets can be of three types: 1. Class I safety cabinets: Class I safety cabinet Figure( 2), have an open front with negative pressure ventilation and a HEPA- filtered air exhaust system. These cabinets are designed to protect the user from infectious agent. Class I cabinets are not suitable for cell culture operations . HEPA filter Glass panel (decontaminated air) Figure (2): biological safety cabinets class IUQU Micro. Raniah 1429-1430 6
  7. 7. 2. The Class II safety cabinets: a. These are the best all-round biological safety cabinets for HEPA filter general microbiological usage. Figure (3). HEPA filter Glass panel (decontaminated air) Figure (3): biological safety cabinets class II b. class II cabinets provide protection to the user, the environment, and the culture by means of a recirculating HEPA-filtered vertical airflow, and HEPA-filtered exhaust air. 3. The Class III cabinets: Ventilated cabinet - totally enclosed. It provides both personnel and specimen protection .Operations are conducted through attached HEPA filter rubber gloves. Both supply HEPA and exhaust air are HEPA- filter filtered. Glass Gloves (decontaminated air) Figure (4): biological safety cabinets class IIIUQU Micro. Raniah 1429-1430 7
  8. 8. Practical No 2 LIGHT MICROSCOPE1. OBJECTIVES: 1.1. To be familiar with different parts of a compound microscope. 1.2. To be familiar with the major application of light microscope. 1.3. To know the use of each objectives lenses.10X for wet preparation, and 100X for oil immersion. To adjust the light source to optimum depending on the preparation being examined by using the condenser and iris diaphragm.2. BACKGROUNDThe use of microscope in all their various forms in known as microscopy. themicroscope is used in the microbiology laboratory to study microorganism. Using asystem of lenses and illumination sources, it makes a microscopic object visible.Microscopes can magnify an abject from 100-100 times of its original size. The size ofbacteria are always expressed on metric units such as millimeter (mm), micrometer(µm) and nanometer (nm). (1 mm= 1000 µm or 1000,000 nm). Figure (5). Figure ( 5): Range of sizes of major microorganisms, and the range of human eye, light microscope, and electron microscope. UQU Micro. Raniah 1429-1430 8
  9. 9. 2.1. Light microscopePrinciple: to magnify an abject the light microscope uses a system of lenses (objectivesand oculars) to manage the path of light beam that travels between the object beingstudied and the eye.2.2. Application of the light microscope2.2.1.Bright field microscopyIt uses a light source that illuminate the entire specimen field. This method is used toexamine stained preparation and sometimes non-stained.2.2.2.Dark field microscopyIt uses light microscope equipped with a special condenser and objective to brightlyilluminate the microorganism in the specimen against a dark background. This methodis used for the examination of unstained motile living microorganisms e.g. Treponemaspecies.2.2.3.Fluorescent microscopyUltraviolet lamp is used instead of ordinary light bulb. The specimen is stained withfluorescent dye that absorbs the energy of short light waves (ultraviolet). The dye thenreleased or emits light of long wavelength such as green light (fluorescence).Commonly used fluorescent dyes are acridine orange, auramine/ rhodamine, andcalcoflour white.2.2.4. Phase contrast microscopyIt uses a modified light microscope that permits greater contrast between substances ofdifferent thickness or density. A special condenser and objective controls theillumination. The result is an image of structure with differing degrees of brightness ordarkness, collectively called contrast. The denser materials appear bright and the partof cell that have a density close to water will appear dark. 3. MATERIALS 1. Compound microscope. 2. Lens cleaning paper/cloth. 3. Immersion oil. 4. Stained preparation. UQU Micro. Raniah 1429-1430 9
  10. 10. 4. METHODS Study different parts of a compound microscope figure (6) and how to start focusing the slide under the microscope figure (7).Major parts of compound microscope are: a. Eye piece (ocular lens): a magnifying lens with magnification power of 10X. b. Body tube: Contains mirrors and prisms that transmit the image from the objective lens to the ocular lens. c. Objective lenses: Primary lenses that magnify a specimen • (10X) Low power objective Low power field (L.P.F), used first before 40X and 100X , to focus the slide on the microscope and bring the image to the ocular lenses. • (40X)High power objective high power field (H.P.F), used secondly after the 10X for examination and wet preparation. • (100X) Oil immersion objective, place a drop of oil is on the slide and then examine it under the 100X objective lens. It used with stained slides. d. Stage: Holds the slide in position e. Condenser: A lens system that condenses light before It passes through the specimen. f. Iris diaphragm: controls the amount of light entering the condenser. g. Coarse and fine adjustment knobs: used for focusing the specimen. Turning the knob changes the distance between the objective lens and the specimen. h. Light: source of illumination, a bulb.4.2.1. Total magnification power The image formed by the objective is enlarged by the ocular lens. The total magnification obtained with any one of the objective lenses is determined by following: Total magnification power= Power of the objective lens X Power of ocular lens e.g., if you are using oil immersion objective (100X), and you know that the power ocular lens is usually 10X. So, the total magnification power =100 X 10 = 1000.4.2.2. Resolving power of a microscope The resolving power of any microscope is a measure of its ability to discriminate between two adjacent objects. The absolute limit of the resolving power is roughly the wavelength of the light used to illuminate the specimen. The wave length of visible light ranges from 400-800 nm.4.2.3. Field of view The circular field you see when you look through the ocular lens. The field of view changes in size at different magnifications.UQU Micro. Raniah 1429-1430 10
  11. 11. Figure (6): Component of light microscopeUQU Micro. Raniah 1429-1430 11
  12. 12. 3. Ad inter djust rpupillar distanc ce. 1. Pllace the specimen slide on the ob bject stage e. 4. Foc cus 5. ad optiimum djust imag contras ge st 2. A the lamb Adjust e brigh htness.Figure (7): How to use the microsc cope at fir (focusi the sp rst ing pecimen slide).UQU Mic cro. Raniah 1429-1430 h 12 2
  13. 13. SAFETY QUIZ : Q.1. what is the importance of hand washing at the beginning and at the end of laboratory work? Q.2. Why we disinfect bench-tops before and after working? Q.3. Why mouth pipetting is not recommended? Q.4. Why used syringes and needles discarded in puncture-proof container? LIGHT MICROSCOPE QUIZ: Q.1. Explain the use of low power, high power, and oil immersion objectives? Q.2. What do you understand by total magnification power of microscope? Q.3. Which objective is used to focus a specimen? Q.4. what is the role of condenser and iris diaphragm in focusing specimen? Q.5. what do you understand by coarse and fine adjustment? Q.6. Which objective are used to examine wet and stained preparation ?UQU Micro. Raniah 1429-1430 13
  14. 14. PRACTICAL No.2 MORPHOLOGY OF BACTERIA1. OBJECTIVES 1.1. To define the morphological types and arrangements of bacteria 1.2. To differentiate between cocci, bacilli, and spirochaetes. 1.3. To identify different size of bacteria. 1.4. To draw the morphological types and arrangements of bacteria.2. BACKGROUND The word morphology means the study of form and structure. Bacteria have a wide variety of size and shapes. The bacteria that posses cell walls exist in three distinct basic morphologic forms. 2.1. Basic forms: 2.1.1. Cocci: spherical forms arranged in pairs (diplococcic), in chains of varying length, and in packet of fours or in groups (clusters). Figure (8) 2.1.2. Bacilli: Rod shaped bacteria may be arranged in pairs , in chains or have irregular arrangement. 2.1.3. Spiral bacteria (Spirilla): the bacteria that appear snake like having a series of rigid curves. 2.1.4. Other forms: Spirochaetes: these are SPIRAL bacteria with a series of flexuous curves. The number of, the depth, and the arrangement of curves vary from one species to another. Vibrios: curved BACILLI arranged irregularly or In pairs. Figure (8). Filamentous bacteria: long thin bacilli, which may show branching. 2.2. Size Bacteria are small and measured in terms of microns (1µ = 1/1000 of a millimeter).Typical bacteria are of 1 µm in diameter, but also vary e.g., anthrax bacillus 4 to 8 by 1 to 1.5 µm and the whooping cough bacillus 1.5 to 1.8 µm by 0.3 – 0.5 µm.3. MATERIALS UQU Micro. Raniah 1429-1430 14
  15. 15. 1. Microscope. 2. Immersion oil. 3. Lens cleaning paper. 4. 6 Stained slide of: 4.1, 4.2, and 4.3 as follow: 4.1.Cocci: o Cocci in pairs diplo- cocci e.g. Neisseria gonorrhea Fig 9-1 o Cocci in chains strepto- cocci e.g. Streptococcus pyogenes Fig 9-2 o Cocci in cluster staphylo- cocci e.g. Staphylococcus aureus Fig 9-3 4.2. Bacilli o Bacilli in pairs. o Bacilli in irregular arrangement. e.g. Escherichia coli Fig 9-4 o Bacilli in chain. e.g. Bacillus cereus Fig 9-5 4.3. Spirochaetes e.g. Treponema pallidum Fig 9-64. METHOD Examine the stained slides provided and draw illustrative labeled diagram on theresults sheet provided, by consulting Figure (8,9). Figure (8): Basic bacterial morphology. UQU Micro. Raniah 1429-1430 15
  16. 16. Figur re(9-1): Co in pair = diploc occi rs coccus, ure(9-2):Co Figu occi in cha ains=strepptococcus s, Neiss seria gonor rrhea .Ligh microsc ht cope 100X. Strep pyogenes. Light mic ptococcus p croscope 100X X. re(9-3):Coc in clus Figur cci ster = stap pylococci, ure(9-4):Ba Figu acilli in irr regular ar rrangemen n hylococcus aureus .Light micro Staph oscope 100 0X. herichia co .Light microscope 100X. Esch oli m e(9-5): Bac in cha Bacillu cereus Figure cilli ain. us ure(9-6): S Figu Spirochaetes. Trepo onema .Light microsco 100X. t ope lidum .Lig micros pall ght scope 100XX.UQU Mic cro. Raniah 1429-1430 h 16 6
  17. 17. 5. RESULTS 5.1. Cocci: Slide Morphology Arrangement Drawing Example In pairs 1 Cocci (diplo-) In chains (strepto-) 2 Cocci In cluster (stapylo-) 3 Cocci 5.2. Bacilli: Slide Morphology Arrangement Drawing Example Random Bacilli (irregular 4 arrangement) 5 Bacilli Chains 5.3. Bacilli: Slide Morphology Drawing Example Spirochaetes 6 (Spiral bacteria ) Students name:__________________________________________ Student No.______________ UQU Micro. Raniah 1429-1430 17