Lab 1 Safety and Lab Guidelines Syllabus Expectations –12 quizzes – drop 1 – no make up quizzes/exercises –Due date – turn in at beginning of lab – Student preparation for lab – Lab reports and questions – study tool 18/18/12MDufilho

Safety and Lab Guidelines Read pp. 1 – 6 Treat all microorganisms (bacteria, viruses, fungi) and chemicals as if they are hazardous Learn and practice the safest level of standard at all times –No food, drinks in the lab EVER –Wash hands thoroughly, after handling microbes, before leaving lab, after removing gloves –Take nothing out of the lab 28/18/12MDufilho

Safety and Lab Guidelines –Open toed shoes –Tie back long hair –Gloves while handling microbes/staining –Antiseptic if exposed –Turn off Bunsen burner when not in use –Eyewash –Fire extinguisher –Keep work area clear 38/18/12MDufilho

Spills and Disposal of Materials Spills and broken glass –Notify instructor immediately before attempting to clean up Disposal of Materials –Broken glass – special container –Biohazard Bag – for contaminated paper towels, gloves, etc. –Test tubes – remove labels & place in racks –Plates – place in disposal bucket –Live cultures on slides – decontaminate/clean –Stained slides – clean and return to slide box 48/18/12MDufilho

Work Area Keep work area free of clutter – do not keep books/papers on work area Wipe with 70% ethanol before and after lab All tubes must be in racks – do not lay tubes on table Always put paper towel on work area and soak it with 70% ethanol 58/18/12MDufilho

8/18/12MDufilho6

Basic Growth Media Lecture text pp What is growth media Why do microbiologists need growth media? What are the requirements for a growth media? 78/18/12MDufilho

Categories of Media Physical States Liquid Semisolid Solid (can be liquefied) Solid (cannot be liquefied) 8 Chemical composition Defined (synthetic, chemically defined) Complex ( non-synthetic, not chemically defined) 8/18/12MDufilho

9 Physical States of Media Liquid – broth; does not solidify Semisolid – contains solidifying agent Solid – firm surface for colony formation –Contains solidifying agent –Liquefiable and nonliquefiable 8/18/12MDufilho

Figure 6.11 Slant tube containing solid media Slant Butt

11 Chemical Content of Media Synthetic – contains pure organic and inorganic compounds in an exact chemical formula Complex or nonsynthetic – contains at least one ingredient that is not chemically definable General purpose media – grows a broad range of microbes, usually nonsynthetic Enriched media – contains complex organic substances such as blood, serum, hemoglobin, or special growth factors required by fastidious microbes 8/18/12MDufilho

Types of Media Culture Media –Majority of prokaryotes have not been grown in culture medium –Six types of general culture media Defined media Complex media Selective media Differential media Anaerobic media Transport media 8/18/1212MDufilho

Figure 6.12 An example of the use of a selective medium Fungal colonies Bacterial colonies pH 7.3pH 5.6

Figure 6.13 The use of blood agar as a differential medium Beta-hemolysis Alpha-hemolysis No hemolysis (gamme-hemolysis)

Figure 6.14 The use of carbohydrate utilization tubes as differential media No fermentation Acid fermentation with gas Durham tube (inverted tube to trap gas)

Figure 6.15 Use of MacConkey agar as a selective and differential medium-overview

Figure 6.16 An anaerobic culture system Clamp Chamber Petri plates Airtight lid Envelope containing chemicals to release CO 2 and H 2 Palladium pellets to catalyze reaction removing O 2 Methylene blue (anaerobic indicator)

18 Most commonly used media –Nutrient broth – liquid medium containing beef extract and peptone –Nutrient agar – solid media containing beef extract, peptone, and agar –Typticase Soy Broth or Agar – beef extract and soy extract –Brain Heart Infusion Broth and Agar – extract of beef heart and brain 8/18/12MDufilho

19 Agar Most commonly used solidifying agent is agar A complex polysaccharide isolated from red algae –Solid at room temperature, liquefies at boiling (100 o C), does not re-solidify until it cools to 42 o C –Provides framework to hold moisture and nutrients –Not digestible for most microbes 8/18/12MDufilho

Exercise 1.3 – Media Prep Important points –Careful measuring of materials –Careful distribution –Sterilization and verification –Storage 208/18/12MDufilho

Weighing Ingredients 218/18/12MDufilho

Mixing the Medium 228/18/12MDufilho

Dispensing the Medium 238/18/12MDufilho

Autoclaving the Media 248/18/12MDufilho

Tubed Media 258/18/12MDufilho

Pouring Agar Plates 268/18/12MDufilho

Ex. 2.1 Ubiquity of Microorganisms Ubiquity? Work in groups of 2 -3 Growth media – Nutrient Agar Label bottom of plates with #, initials & date Invert – why? Tape plates together Incubation T – 25 o and 37 o 278/18/12MDufilho