Introduction to Microbiology and Laboratory Safety Biosafety

Media Types General Purpose Media Enriched Media Selective Media Differential Media

Media Types General Purpose Media: Supports the growth of many microorganisms i.e. Luria Agar Enriched Media: Has special nutrients to encourage the growth of fastidious heterotrophs i.e. Blood Agar Selective Media: Favors the growth of one type of microorganisms and inhibits the growth of others Luria + penicillin Agar Differential Media: Distinguishes between different groups of bacteria on the basis of biochemical characteristics i.e. Eosin Methylene Blue Agar

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 identification kits autoclave Clorox bleach, like you buy at the supermarket, diluted to 5-10% is the best cleaning agent for labs.

Microorganism Isolation Techniques Using an Inoculating Loop Streaking Methods

How to hold an Inoculating Loop

Streaking and Flaming Procedure Flame the loop to sterilize it and let cool. Position the plate so that the spot of inoculum is nearest the hand not holding the loop (the opposite hand). Lift the plate lid with the opposite hand; just enough to get the loop inside and touch the loop to the inoculum spot. It is often helpful to treat the inoculating loop as if it were a pencil - steadying the loop by resting the heel of the hand against the lab bench. Move the loop back and forth across the spot and then gradually continue toward the center of the plate as you sweep back and forth. Use a very gentle and even pressure. When creating each phase, do not worry about keeping each pass across the plate separate from previous ones. When about 30% of the plate has been covered by the first streaking phase, remove the loop and flame sterilize it. Repeat the above procedure for the second phase, but this time pick up some inoculum by crossing into the first phase 2-3 times and then not passing into it again (Figure 1-5). Repeat as necessary for the third and fourth phases. After streaking the plate, flame sterilize the loop before setting it down.

Triple Streak Method

Streak Plate http://www. sumanasinc

Streak plate method of isolation For MICR 2909 Lecture 2, 2001 Streak plate method of isolation 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. BSc(MolBiol) Lect 2.ppt

Procedure for Making a ‘Smear’ Using aseptic technique remove a colony from a plate or cells from your slant.  Be carefully to gently touch the surface of your culture with the inoculating loop. Make a circular motion in the middle of the circle to spread the cells equally in this region of the slide Add a drop of water in the middle Mix again Let Air dry Run the slide through the flame until the slide is warm ( The frosted side should be down)  This fixes the bacteria to the slide Let the slide cool Place in the metal tray or in the rack

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 base( 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.

Flaming tubes

Transferring Microorganisms to Slant Test Tubes

Streaking a slant

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( water) Flame mouth of culture tube and close Open culture tube with broth( should be labeled) Dip loop into new broth and mix Flame mouth of tube and close Flame loop Place to the side of your rack

Identifying Bacteria Cultures:

Colony Morphology

Colony Morphology Colony morphology Color Shape Margin Elevation

Stains and Staining Bacteria are slightly negatively charged at pH 7.0 For MICR 2909 Lecture 2, 2001 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 BSc(MolBiol) Lect 2.ppt

Procedure for Simple Stains For MICR 2909 Lecture 2, 2001 Procedure for Simple Stains BSc(MolBiol) Lect 2.ppt

Differential Stains Gram stain Crystal violet: primary stain For MICR 2909 Lecture 2, 2001 Differential Stains Gram stain Crystal violet: primary stain Iodine: mordant Alcohol or acetone-alcohol: Safranin decolourizer : counterstain Gram positive: purple Gram negative: pink-red Staphylococcus aureus Escherichia coli BSc(MolBiol) Lect 2.ppt

Differential Stains Acid-fast stain For MICR 2909 Lecture 2, 2001 Differential Stains Acid-fast stain Used to detect Mycobacterium species Acid fast stain Acid fast stain (Ziehl-Neelson) for identifying mycobacteria The lipid mycolic acid (from mycobacteria) is the determinant of retaining the basic fuchsin in the acid-fast stain BSc(MolBiol) Lect 2.ppt

Procedure for Gram Stain All staining work is to be done at the sink Care should be taken to work directly over the sink Place 1 drop of crystal violet stain on the smear ( 1 minute) Rock or roll the slide to cover the area Use the water bottle to drip water down the slide Place 1 drop of iodine on the slide ( 1 minute) Place 1 drop of alcohol on the slide 10 seconds ( KEY – do not leave on longer than 10 seconds or it will decolorize) Place 1 drop of saffranin on the slide for 1 minute Rinse with water from the bottle Let the slide air dry  

Streptococcus

Staphylococcus aureus

Gram negative bacilli

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

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

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.

Guidelines for Microorganism Use The microbes are placed into 4 categories called : 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.

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.

Biosafety Levels for Infectious Agents BSL Agents 1 Not known to consistently cause disease in healthy adults 2 Associated with human disease, hazard = percutaneous injury, ingestion, mucous membrane exposure 3 Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences 4 Dangerous/exotic agents which pose high risk of life-threatening disease, aerosol-transmitted lab infections; or related agents with unknown risk of transmission This table identifies the types of microorganisms typically placed in the four biosafety levels.

Recommended Biosafety Level Practices* BSL Practice 1 Standard Microbiological Practices 2 BSL-1 practice plus: Limited access, Biohazard warning signs, "Sharps" precautions, Biosafety manual defining any needed waste decontamination or medical surveillance policies 3 BSL-2 practice plus: Controlled access, Decontamination of all waste, Decontamination of lab clothing before laundering, Baseline serum antibody analysis 4 BSL-3 practices plus: Clothing change before entering, Shower on exit, All material decontaminated on exit from facility 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.

Engineering Controls by Biosafety Level BSL Safety Equipment (Primary Barriers) Facilities (Secondary Barriers) 1 None required Open bench top & sink required 2 Primary barriers = Class I or II BioSafety Cabinets; laboratory coats; gloves; face protection as needed BSL-1 plus: • Autoclave available 3 Primary barriers = Class I or II BioSafety Cabinets; protective lab clothing; gloves; respiratory protection as needed BSL-2 plus: • Self-closing, double-door access • Exhausted air not recirculated • Negative airflow into laboratory 4 Primary barriers = Class III BioSafety Cabinets or in combination with full-body, air-supplied, positive pressure suit BSL-3 plus: • Separate building or zone • Dedicated supply and exhaust, vacuum, and decon systems This table identifies the engineering controls (safety equipment and contamination barriers) required for sfe use of microorganisms at each biosafety level.

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

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 Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments or work with cultures and specimens are in progress. A biohazard sign can be posted at the entrance to the laboratory whenever infectious agents are present. The sign may include the name of the agent(s) in use and the name and phone number of the investigator. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in the work areas. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only. 2.3

Biosafety Level 1 Standard Microbiological Practices What can be done in the absence of handwashing facilities? Use antiseptic hand cleaner and clean towels. Persons wash their hands after they handle viable materials, after removing gloves, and before leaving the laboratory. 2.3

Standard practices also include: Keep work areas uncluttered and clean No food in lab refrigerator Minimize splashes and aerosols Decontaminate work surfaces daily Maintain insect & rodent control program

Decontamination Sterilization Disinfection

Decontamination Definition Sterilization The use of a physical or chemical procedure to destroy all microbial life, including large numbers of highly resistant bacterial spores.

Disinfection Definition 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.

Decontamination Methods Heat Chemical Radiation

Decontamination Heat Types Moist – steam Dry Incineration *The most effective method of sterilization

Decontamination Chemical Types Liquids, i.e. chlorox, hydrogen peroxide Gases, i.e. ethylene oxide

Decontamination Chemical 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

In case of a spill 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

Aseptic Technique First requirement for study of microbes For MICR 2909 Lecture 2, 2001 Aseptic Technique First requirement for study of microbes pure cultures, free of other microbes Maintain a clean environment; work close to the flame Aseptic Transfers Purpose Transfer of microbiological cultures from one medium to another sterile medium without contamination of the culture, sterile medium, or the surroundings.   Principle Certain techniques are necessary to handle tubes of media, plated media, and inoculating loops or swabs. Practice in the manipulations, while maintaining aseptic conditions are known as "aseptic techniques." Refer to your laboratory text for acceptable methods of transferring microbial cultures. However, practice is the only way to master the techniques. Additional Information 1. Gather all the necessary materials (bacterial stock culture, growth media, bunsen burner, transfer tools). 2. Label the tubes properly, following the outlined instructions. 3. Check each organism to be transferred, using the stock cultures. 4. Practice adjusting the flame of the Bunsen burner. 5. Discard contaminated materials properly, referring to the guidelines given in class. Sterilize the loop or needle by holding in the flame of a Bunsen burner. The metal must glow red before sterilization is considered complete. When transferring organisms, it is important to maintain a clean environment. Talking, coughing or sneezing should be avoided when performing bacterial transfers from one media to another. Avoid drafts. Maintain a clean environment by disinfecting tabletops before and after working with microorganisms. Common surface disinfectants are 70% Ethanol and 10% Lysol. BSc(MolBiol) Lect 2.ppt

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