Introduction to Microbiology and Laboratory Safety

1. Introduction to Microbiology and Laboratory Safety

2. Introduction to Microbiology and Lab Safety

3. Microbiology Techniques

4. Safety NO FOOD OR DRINKS! Wash hands thoroughly Disinfect counters and work area Tie hair back Smock, apron, or lab coat optional Gloves and goggles optional Closed toed shoes required Eyewash in middle back sink Fire blankets in back on shelf by the door Fire extinguishers in hallway

5. Safety Resources A number of safety references are available (CDC, WHO, ISO). More detail of what is discussed in this presentation can be found in any of these references. Details on: Safety Responsibilities Management Staff Facility Management Design Identification of hazards Housekeeping practices Biologic Safety Chemical Safety This presentation focuses primarly on biosafety A number of safety references are available (CDC, WHO, ISO). More detail of what is discussed in this presentation can be found in any of these references. Details on: Safety Responsibilities Management Staff Facility Management Design Identification of hazards Housekeeping practices Biologic Safety Chemical Safety This presentation focuses primarly on biosafety

6. Biosafety Level 1 - Standard Microbiological Practices Restrict or limit access when working Prohibit eating, drinking and smoking in the laboratory Pipetting by mouth strictly forbidden

7. Biosafety Level 1 - Standard Microbiological Practices What can be done in the absence of handwashing facilities? Use antiseptic hand cleaner and clean towels. What can be done in the absence of handwashing facilities? Use antiseptic hand cleaner and clean towels.

8. Always thoroughly wash hands upon entering and leaving lab Keep work areas uncluttered and clean No food in lab refrigerator Minimize splashes and aerosols Disinfect work surfaces daily Maintain insect & rodent control program Biosafety Level 1 - Standard Microbiological Practices

9. Decontamination Sterilization Disinfection

10. General Lab Use - Hypochlorite Solutions Large Spills/Large Organic Load undiluted from bottle Small Spills/Virus Inactivation 10% - 1:9 General Surface Disinfection 1% - 1:99 Chemical Decontamination

11. Disinfection: The use of a physical or chemical procedure to virtually eliminate all recognized pathogenic microorganisms but not all microbial forms (bacterial endospores) on inanimate objects. Disinfection

12. Decontamination Sterilization: The use of physical or chemical procedures that destroy all microbial life forms, including highly resistant bacterial endospores. Autoclave: Pressurized steam at 15 psi and 121oC for an average of 20 min (10 � 40 min depending on bulk and load)

13. In case of a spill Get the instructor, do not pick up glass! Wear disposable gloves Cover large blood spill with paper towels and soak with 1% (10000 ppm) of household bleach and allow to stand for at least 5 minutes Small spill - wipe with paper towel soaked in 1% bleach Discard contaminated towels in infective waste containers Wipe down the area with clean towels soaked in a same dilution of household bleach

14. Microbiology Lab Equipment Microscope (with accessories) Inoculation loops Source of flame (Bunsen burner) Microscope slides and Cover slips Gram staining kits (can purchase from science supply store) Petri dishes and proper growth media Incubators Autoclave Clorox bleach, like you buy at the supermarket, diluted to 5-10% or disinfectant provided in lab.

15. Microscopy: The Instruments In a compound microscope the image from the objective lens is magnified again by the ocular lens. Total magnification =objective lens ? ocular lens

16. Microscopy Lower stage completely Rotate nosepiece to 4x objective Find area of specimen on slide with naked eye (you may want to circle the specimen with a sharpie) Place specimen over circle of light (adjust to area of interest using stage dials) Crank stage all the way up using coarse (large knob) focus while looking in ocular (specimen should come into focus) Move 4 and 20x objectives out of the way Place oil directly on slide and rotate objective labeled with �oil, black line or 40x.� Use ONLY fine focus (small knob) only to bring into view through ocular

17. Aseptic Technique Required for all microbiology preparations to assure that contaminants are not introduced. On a personal note, aseptic technique assures that infectious agents are not spread to you, fellow students, or the laboratory surfaces.

18. general rules of microbiology laboratory The inoculating loop is usually used for making transfers of bacterial cultures (see next few slides for technique). Allow the loop to cool sufficiently so that any organisms to be tested will not be killed by the hot wire, but do not allow the loop to contact anything during the cooling period or contamination will result. Learn to remove and replace the caps or lids efficiently without setting them on the countertop or leaving the cover off too long. After the transfer is completed the loop must be sterilized again. Follow the procedure outlined on the following slides to prevent splattering of infectious materials. It is probably easier to work while sitting down. Attention to details and practice will allow you to work both rapidly and accurately.

19. How to hold an Inoculating Loop

20. FLAMING A LOOP

21. FLAMING A LOOP

22. FLAMING A LOOP

23. Smear preparation Label top side of slide with small piece of masking tape (or anything that won�t wash off) Apply a loop full of wall Obtain bacteria specimen aseptically ALLOW SMEAR TO COMPLETELY AIR DRY (DO NOT TRY TO SPEED UP DRYING BY HEATING!) After slide has completely air-dried then gently heat fix in flame by hold slide with a clothes pin and quickly waving bottom side of slide over flame 2 � 3 times.

24. Stains and Staining Bacteria are slightly negatively charged at pH 7.0 Basic dye stains bacteria Acidic dye stains background Simple stain Aqueous or alcohol solution of single basic dye

25. Procedure for Simple Stains

26. Simple stain Prepare slide and air dry (circle specimen on bottom of slide with sharpie to enable finding specimen when you put it on the scope) Heat fix Apply methylene blue Let stand for 60 sec Rinse with water Dry slide

27. Differential Stains Gram stain Crystal violet: primary stain Iodine: mordant Alcohol or acetone-alcohol: decolorizer Safranin: counterstain Gram positive: purple Gram negative: pink-red

28. Procedure for Gram Stain All staining work is to be done over the sink or trough with light even flow of running water! Cover specimen on smear with of crystal violet stain (1 minute) Rock or roll the slide to cover the area Rinse completely with water using gentle flow from squirt bottle Cover specimen with iodine on the slide (1 minute) Rinse with water Place 1 drop of alcohol on the slide 10 seconds (KEY � do not leave on longer than 10 seconds or it will over decolorize) Rinse with water Cover specimen with saffranin (1 minute ) Rinse with water from the bottle Let the slide air and/or blot dry

29. Streptococcus spp.

30. Staphylococcus aureus

31. Gram negative bacilli

32. CULTIVATION AND ISOLATION OF BACTERIA Diagnostic bacteriology is concerned with the isolation and identification of bacteria in a specimen from a patient. These specimens, unless from a normally sterile site of the body, rarely contain a single bacterial type, but are mixtures of the disease-producing bacteria and the host's normal or indigenous flora. Since accurate studies of the biochemical and the antigenic properties of a bacterial species are possible only through the use of pure cultures, it is necessary to have a reliable and rapid method that will permit the isolation of possible pathogenic organisms. An inoculum from the specimen is streaked on solid agar in a manner, which physically separates most of the bacterial types, permitting them to form discrete colonies. This procedure is facilitated whenever possible by the use of either a selective medium that inhibits the growth of species not sought or by the use of a differential medium, which imparts a recognizable appearance to the colonies of the type sought. Upon transfer of this mixed colony to a medium without the inhibitors, both types of bacteria may grow, and a pure culture will not be obtained. Consequently, it is often necessary to streak a second plate of the same selective medium with a colony from the first selective plate in order to obtain a pure culture of the bacterial species that you are attempting to isolate.

33. COLONY ISOLATION Four quadrant technique: Using a sterile loop, streak cultures (liquid broth or isolated colonies picked from plates) over one-fourth of the surface of an agar plate. Then flame the loop again. Air cool a flamed loop or cool it by touching an unstreaked area of agar on the same plate. Pass the cooled loop three or four times over the initial streaked portion of the plate. Streak it, without overlap, to the next quadrant.

34. COLONY ISOLATION Flame the loop and allow it to cool as described above in Step 2. Pass the loop over the streaked portion of the second quadrant two or three times and then streak the material without overlapping over the third quadrant of the plate. Repeat Step 5 to streak the last quadrant. � �

35. COLONY ISOLATION

36. Streak Plate

37. Streak plate Good and bad At all costs, there must be prevention of contamination. Often use cotton wool stoppers to flasks, tubes. Petri dish: ideal for solid medium and allows gaseous diffusion without dust Development of solid media needed for colony formation initially used surface of freshly cut vegetables eg potato gelatin (1881) used low melting point (< 37oC) protein, therefore a nutrient agar-agar (Hess, Koch�s lab) polysaccharide - nutritionally inert melts 100oC ; solidifies ~40oC Streak Plate Method of Isolation Purpose The streak plate technique is the most widely used method of obtaining isolated colonies from a mix of cultures. � Principle The streak plate technique is essentially a method to dilute the number of organisms, decreasing the density. This allows for individual colonies to be isolated from other colonies. Each colony is considered "pure," since theoretically, the colony began with an individual cell. �Additional Information (see also p. 53 in the lab text for diagrams.)1. Begin with inoculating the first, or primary, quadrant of the agar plate. Use a light touch. Don't penetrate or scrape the agar surface. Cover plate with lid. 2. Flame the loop, cool by touching an uninoculated portion of the surface. 3. Now rotate the plate. Open lid and streak again, following the diagram in the exercise book. Remember: you are picking up growth from quadrant one, and using this as your inoculum for quadrant two. 4. Flame loop; rotate plate, and repeat procedure for quadrants three and four. The proper wrist action and light touch takes practice. At all costs, there must be prevention of contamination. Often use cotton wool stoppers to flasks, tubes. Petri dish: ideal for solid medium and allows gaseous diffusion without dust Development of solid media needed for colony formation initially used surface of freshly cut vegetables eg potato gelatin (1881) used low melting point (< 37oC) protein, therefore a nutrient agar-agar (Hess, Koch�s lab) polysaccharide - nutritionally inert melts 100oC ; solidifies ~40oC Streak Plate Method of Isolation Purpose The streak plate technique is the most widely used method of obtaining isolated colonies from a mix of cultures. � Principle The streak plate technique is essentially a method to dilute the number of organisms, decreasing the density. This allows for individual colonies to be isolated from other colonies. Each colony is considered "pure," since theoretically, the colony began with an individual cell. �Additional Information (see also p. 53 in the lab text for diagrams.)1. Begin with inoculating the first, or primary, quadrant of the agar plate. Use a light touch. Don't penetrate or scrape the agar surface. Cover plate with lid. 2. Flame the loop, cool by touching an uninoculated portion of the surface. 3. Now rotate the plate. Open lid and streak again, following the diagram in the exercise book. Remember: you are picking up growth from quadrant one, and using this as your inoculum for quadrant two. 4. Flame loop; rotate plate, and repeat procedure for quadrants three and four. The proper wrist action and light touch takes practice.

38. Procedure for Making a Smear Place a very small drop of water on your clean slide. Using aseptic technique remove a colony from a plate or cells from your slant.� Be careful to just gently touch the surface of your culture with the inoculating loop. Transfer inoculum to center of water drop on slide. Mix in a circular motion to equally spread specimen to the size of about a dime. You must allow specimen to then air dry! This may take a while (5-20 min). You will ruin your smear if you try to accelerate drying by rapidly heating an in a flame. Now wave the slide through a flame 3-4 time. This fixes the bacteria to the slide Let the slide cool Place in the metal tray or in the rack

39. Procedure for Transferring Microorganisms to a Slant 1. Wrap fingers of non dominant hand around the culture tube containing broth for transfer 2. Using the pinkie finger of your dominant hand twist the red cap from the tube.� Hold in your pinkie and do not place it on the counter 3.���� Pass the mouth of the culture tube across the flame 4.���� Direct the inoculating needle into the broth. 5.���� Flame the mouth of your broth culture tube and replace the cap.� Place it in your rack . 6.���� Pick up the slant in your non dominant hand 7.���� Twist off the red cap 8.���� Flame the mouth of the slant tube 9.���� Direct the inoculating needle into the tube and � stab� the agar in the butt 10. Withdraw on the entry line and when you reach the surface make a simple streak along the face. 11.� Flame the mouth of the tube and replace the cap 12. Flame your inoculating needle and replace in your rack

40. Flaming tubes

41. Transferring Microorganisms to Slant Test Tubes

42. Streaking a slant

43. Procedure for Transferring Microorganisms to Broth Test Tubes Steps for Transfer of Broth to Broth Hold loop or needle with dominant hand( right ) Flame the loop Hold culture tube in left hand Remove red cap with pinkie of right hand Flame mouth of culture tube� Place loop into broth Flame mouth of culture tube and close Open culture tube with broth Dip loop into new broth and mix Flame mouth of tube and close Flame loop Place to the side of your rack

44. Identifying Bacteria Cultures

45. Colony Morphology Colony morphology Color Shape Margin Elevation

46. Colony Morphology

47. Colony morphology

52. Media Types General purpose Enriched Selective Differential

53. Media Types General purpose: Supports growth of most non fastidious organisms Nutrient and TSA Enriched fastidious organisms Streptococcus pyogenes, Neisseria gonorrheae Blood and chooclate Selective: Favors the growth of one type of microorganisms and inhibits the growth of others Saboraud, EMB, MSA, SS Differential Media: Distinguishes between different groups of bacteria on the basis of biochemical characteristics EMB, MacConkey, HE, SS

54. Safety in the Microbiology Lab An Introduction to Principles and Practices at Biosafety Levels 1, 2, 3, & 4

55. Microorganism Categories How are microorganisms categorized? By genetics to show how they are related By tissues they infect to show how they cause disease By pathogenicity and communicability (also known as their BioSafety Level) Microorganisms can be categorized by a number of techniques. Examples include: Phylogenetically to show genetic relatedness Anatomically, that is by body system, to relate infectious disease processes By their ability to cause disease and be transmitted person to person Microorganisms can be categorized by a number of techniques. Examples include: Phylogenetically to show genetic relatedness Anatomically, that is by body system, to relate infectious disease processes By their ability to cause disease and be transmitted person to person

56. Guidelines for Microorganism Use Besides federal law and regulations other guidelines exist for the use and control of microorganisms: CDC/NIH Biosafety in Microbiological and Biomedical Laboratories (BMBL) WHO (World Health Organization) Biosafety Manual USDA (United States Department of Agriculture) protocols National and international agencies have defined regulations for the control and use of microorganisms. Several detailed references have been published and are available from these agencies.National and international agencies have defined regulations for the control and use of microorganisms. Several detailed references have been published and are available from these agencies.

57. Guidelines for Microorganism Use Microbes placed in 4 categories: Biosafety Levels (BSL 1-4) A common organizational and naming system has been developed whereby microorganisms have identified by their ability to cause disease and be transmitted. This �biosafety level� system has four categories.A common organizational and naming system has been developed whereby microorganisms have identified by their ability to cause disease and be transmitted. This �biosafety level� system has four categories.

58. BSL Labs Microbiology Laboratories are set up and maintained to meet a specific containment level. The designated level conveys information about infection potential and engineering controls implemented to protect workers. Specialized laboratories are required for the safe handling and use of each category of microorganism at there respective biosafety level.Specialized laboratories are required for the safe handling and use of each category of microorganism at there respective biosafety level.

59. Biosafety Levels for Infectious Agents This table identifies the types of microorganisms typically placed in the four biosafety levels. This table identifies the types of microorganisms typically placed in the four biosafety levels.

60. Recommended Biosafety Level Practices This table identifies the practices required at each of the four biosafety levels. Important to all levels are strict observance of standard (good) laboratory practices including: 1. Eating, drinking, the use of cosmetics, gum and tobacco products are strictly prohibited in the lab. Do not manipulate contact lenses in the lab. Tie hair back. Keep hands away from face at all times. Do not put anything (e.g.. pencils) in mouth while in the lab. Protective clothing is recommended while in the lab. Exposed wounds should be covered and protected. 3. Know how to use the emergency eyewash station. In case of fire: Stop, Drop and Roll. Call for help. 4. Disinfect your work space at the beginning and end of lab time. Always wash hands thoroughly before leaving lab. 5. Handle bacterial cultures with extreme care. NEVER pipette by mouth! Learn SPILL PROCEDURE. Cultures NEVER leave the lab. This table identifies the practices required at each of the four biosafety levels. Important to all levels are strict observance of standard (good) laboratory practices including: 1. Eating, drinking, the use of cosmetics, gum and tobacco products are strictly prohibited in the lab. Do not manipulate contact lenses in the lab. Tie hair back. Keep hands away from face at all times. Do not put anything (e.g.. pencils) in mouth while in the lab. Protective clothing is recommended while in the lab. Exposed wounds should be covered and protected. 3. Know how to use the emergency eyewash station. In case of fire: Stop, Drop and Roll. Call for help. 4. Disinfect your work space at the beginning and end of lab time. Always wash hands thoroughly before leaving lab. 5. Handle bacterial cultures with extreme care. NEVER pipette by mouth! Learn SPILL PROCEDURE. Cultures NEVER leave the lab.

61. Engineering Controls by Biosafety Level This table identifies the engineering controls (safety equipment and contamination barriers) required for sfe use of microorganisms at each biosafety level. This table identifies the engineering controls (safety equipment and contamination barriers) required for sfe use of microorganisms at each biosafety level.