1. Introduction to Microbiology and Laboratory Safety
Biosafety

2. Media Types General Purpose Media Enriched Media Selective Media
Differential Media

3. 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

4. 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.

5. Microorganism Isolation Techniques
Using an Inoculating Loop
Streaking Methods

6. How to hold an Inoculating Loop

7. 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.

8. Triple Streak Method

9. Streak Plate http://www. sumanasinc

10. 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

11. 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

12. 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.

13. Flaming tubes

14. Transferring Microorganisms to Slant Test Tubes

15. Streaking a slant

16. 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

17. Identifying Bacteria Cultures:

18. Colony Morphology

19. Colony Morphology
Colony morphology
Color
Shape
Margin
Elevation

20. 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

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

22. 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

23. 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

24. 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

25. Streptococcus

26. Staphylococcus aureus

27. Gram negative bacilli

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

29. 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

30. 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.

31. 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.

32. 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.

33. 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.

34. 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.

35. 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.

36. 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

37. 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

38. 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

39. 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

40. Decontamination
Sterilization
Disinfection

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

42. 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.

43. Decontamination Methods
Heat
Chemical
Radiation

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

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

46. 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

47. 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

48. 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

49. Thank you