Lab 1 Safety and Lab Guidelines Syllabus Expectations 7quizzes – 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 5/29/2018 MDufilho

Safety and Lab Guidelines Read pp. 1 – 11 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 5/29/2018 MDufilho

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 5/29/2018 MDufilho

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 5/29/2018 MDufilho

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 5/29/2018 MDufilho

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Basic Growth Media Lecture text pp. 178- 182 What is growth media Why do microbiologists need growth media? What are the requirements for a growth media? Nutrients? Physical requirements Temperature 5/29/2018 MDufilho

Figure 6.4 The effects of temperature on microbial growth. Minimum Maximum Optimum 5/29/2018 22ºC MDufilho 30ºC 37ºC

Culturing Microorganisms Culture Media Majority of prokaryotes have not been grown in culture medium Nutrient broth is common medium Agar is a common addition to many media Complex polysaccharide derived from certain red algae Produces a solid surface for colonial growth Most microbes cannot digest agar 5/29/2018 MDufilho

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 5/29/2018 MDufilho

Culturing Microorganisms Culture Media Six types of general culture media Defined media Complex media Selective media Differential media Anaerobic media Transport media 5/29/2018 MDufilho

Culturing Microorganisms Culture Media Defined media Medium in which the exact chemical composition is known Fastidious organisms require the addition of a large number of growth factors 5/29/2018 MDufilho

Culturing Microorganisms Culture Media Complex media Exact chemical composition is unknown Contain nutrients released by partial digestion of yeast, beef, soy, or proteins Support growth of wide variety of microorganisms Used to culture organisms with unknown nutritional needs 5/29/2018 MDufilho

Figure 6.12 An example of the use of a selective medium. Bacterial colonies Fungal colonies pH 7.3 pH 5.6 5/29/2018 MDufilho

Culturing Microorganisms Culture Media Enrichment media Use of a selective medium to increase the numbers of a chosen microbe to observable levels May require a series of cultures to enrich for the desired microbe Cold enrichment used to enrich a culture with cold-tolerant species 5/29/2018 MDufilho

Figure 6.13 The use of blood agar as a differential medium. Beta-hemolysis Alpha-hemolysis No hemolysis (gamma-hemolysis) 5/29/2018 MDufilho

Acid fermentation with gas Figure 6.14 The use of carbohydrate utilization tubes as differential media. Durham tube (inverted tube to trap gas) 5/29/2018 No fermentation MDufilho Acid fermentation with gas

serotype Choleraesuis Figure 6.15 The use of MacConkey agar as a selective and differential medium. Escherichia coli Escherichia coli Escherichia coli Staphylococcus aureus Staphylococcus aureus (no growth) Salmonella enterica serotype Choleraesuis Nutrient agar MacConkey agar MacConkey agar 5/29/2018 MDufilho

Culturing Microorganisms Culture Media Anaerobic media Obligate anaerobes must be cultured in the absence of free oxygen Reducing media contain compounds that combine with free oxygen and remove it from the medium Petri plates are incubated in anaerobic culture vessels Sealable containers that contain reducing chemicals 5/29/2018 MDufilho

Figure 6.16 An anaerobic culture system. Clamp Airtight lid Chamber Palladium pellets to catalyze reaction removing O2 Envelope containing chemicals to release CO2 and H2 Methylene blue (anaerobic indicator) Petri plates 5/29/2018 MDufilho

Culturing Microorganisms Culture Media Transport media Used by hospital personnel to ensure clinical specimens are not contaminated and to protect people from infection Rapid transport of samples is important 5/29/2018 MDufilho

Most commonly used media Nutrient broth – liquid medium containing beef extract and peptone Nutrient agar – solid media containing beef extract, peptone, and agar Trypticase Soy Broth or Agar – beef extract and soy extract Brain Heart Infusion Broth and Agar – extract of beef heart and brain 5/29/2018 MDufilho

Exercise 1.3 – Media Prep Important points Careful measuring of materials Careful distribution Sterilization and verification Storage 5/29/2018 MDufilho

Weighing Ingredients 5/29/2018 MDufilho

Mixing the Medium 5/29/2018 MDufilho

Dispensing the Medium 5/29/2018 MDufilho

Pouring Agar Plates 5/29/2018 MDufilho

Autoclaving the Media 5/29/2018 MDufilho

Ex. 2.1 Ubiquity of Microorganisms Work in groups of 2 -3 Growth media – Nutrient Agar Label bottom of plates with #, initials & date Invert – why? Tape plates together Incubation T – 25o and 37o Inventory First 5/29/2018 MDufilho