1. Intro to Microbiology Mrs. Cox (Adapted from Techniques in Microbiology, A Student Handbook by John M. Lammert, 2007) 2014-15

2. Safety First! Wash your hands before you begin and after you complete your work. Also, before you leave the lab for any reason. Keep your personal items (coat, cosmetics, backpack, electronic devices) away from the lab bench. Dress appropriately for lab: closed-toed shoes, safety glasses, tie back long hair, wear gloves, and apron. Absolutely no food or beverages (even water) in the lab while working with bacteria. Keep hands away from your mouth and eyes. Do not remove cultures, reagents or other materials from the lab without permission.

3. More Safety and Getting Started Decontaminate your work surface with a disinfectant before you begin and after you complete your work. When using a bunsen burner, position it so that you will not burn yourself, and turn off the gas when the flame is not needed. Keep bacteria cultures in a test tube rack or other holder when working at the bench or walking around the lab. Place all contaminated tubes, plates and waste materials in appropriate receptacles for subsequent sterilization. If anything containing living bacteria spills, cover with a paper towel and disinfectant. Allow to remain on the spill for at least 20 minutes!

4. This is what we are trying to avoid: CONTAMINATION! http://runforsushi.blogspot.com/2011/05/contamination-station.html

5. What is the difference between disinfection and sterilization? Sterilization is the process in which all living cells, spores, and viruses are completely destroyed or removed from an object or environment. Disinfection is the killing, inhibition, or removal of microorganisms that cause disease. Disinfection may not necessarily eliminate spores or all of the microorganisms from an object or environment. From: http://microbiology.mtsinai.on.ca/faq/cleaning.shtmlhttp://microbiology.mtsinai.on.ca/faq/cleaning.shtml

6. The Autoclave The autoclave is essentially a high temperature pressure cooker! It creates moist heat at temperatures high enough to kill bacterial endospores. Steam under pressure creates the necessary 121 o C. The sterilization time for biohazard waste is at least 45 minutes. Materials sterilized in an autoclave must be able to withstand the high temperatures without breaking down or melting. Plastic petri dishes are sterilized in autoclaveable plastic bags.

7. Autoclave Safety Do not operate the autoclave without supervision. Open the door slowly, only an inch at the end of a sterilization cycle. Wait 10 minutes before removing objects from an autoclave. Super heated water could boil after autoclaving if it is even slightly agitated. Wear insulated gloves when unloading hot materials.

8. Adjusting the Bunsen Burner We will use a Bunsen burner to sterilize instruments used at the lab bench, like the inoculating loop, the bacteria spreader, and the mouth of broth tubes. We will light the Bunsen burner with a striker. The hottest point is at the tip of the bright blue cone. We adjust the size of the flame by opening or closing the air holes. http://www.uwplatt.edu/chemep/chem/chemscape/labdocs/catofp/bunsbur/bunsbur2.htm

9. Bacteriology… Bacteria are ALIVE!

10. Bacteria are alive! That means they… REPRODUCE (quickly!) Maintain HOMEOSTASIS (respiration and excrete waste) Contain GENETIC MATERIAL Use RAW MATERIALS for energy CHANGE over time Are made of CELLS GROW and DEVELOP RESPOND to their environment

11. So…you need to know your bacteria’s ideal growing condidtions: Is it AEROBIC, ANAEROBIC or FACULTATIVE? What does FACULTATIVE mean? We do not have the capability to grow ANAEROBIC bacteria this year…do you need to make a change? What temperature does it thrive in? What are the upper and lower temperature limits? We have a 37 o C incubator. Room temperature is also an option (25 o C). That is all. We should probably begin recording daily temperatures of our incubator to ensure it’s not getting too hot or too cold!

12. What kind of media (food) does your bacteria prefer? Most will be good with basic Nutrient Agar or Broth. It contains all the necessary “food” for most bacteria. Other types of media are either SELECTIVE or DIFFERENTIAL or both. Selective means the media contains an ingredient that selects FOR a certain type of bacteria: MacConkey Agar contains ingredients selects FOR Gram Negative organisms. Differential means the media contains an ingredient that differentiates between two different characteristics of bacteria: MacConkey Agar contains lactose which distinguishes between lactose fermenters and non lactose fermenters

13. The Gram Stain… Is the first, most basic step in identifying your bacteria. It allows you to determine the bacteria’s shape (rod, cocci, etc.) It also breaks all bacteria into two large groups: Gram positive and Gram negative. The gram stain is a multistep process that uses several different stains: Crystal Violet, Gram’s iodine, and Safranin. These stains may or may not be absorbed by the bacteria cell wall, giving the bacteria a distinct purple or red color.

14. Gram Staining… Gram POSITIVE bacteria will be PURPLE in color. Gram NEGATIVE bacteria will be pink-ish RED in color. Some bacteria contain lots of PEPTIDOGLYCAN and others don’t. If they have lots of PEPTIDOGLYCAN, they will be GRAM POSITIVE.

15. Homework Homework: Know your bacteria name, and be sure to research its gram stain characteristics, the optimal growth conditions for your organism and where it is commonly found in nature. You need to know why you chose this bacteria and how it connects to your project.

16. Supplies you will need for bacteriology… Your bactierial culture (order 2!) Pre-poured agar plates…lots of them…probably 5 sleeves of 25-30! Gloves Micropipettor tips (of various sizes) Nutrient broth (pre-made is easiest!) What else? Maybe sterile, blank antibiotic discs, maybe known antibiotic discs (for comparison) A sharpie