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 8/18/12 MDufilho 1

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 8/18/12 – Take nothing out of the lab MDufilho 2

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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 MDufilho 3

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 8/18/12 – Stained slides – clean and return to slide box MDufilho 4

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 8/18/12 MDufilho 5

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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? 8/18/12 MDufilho 7

Categories of Media

• • • •

Physical States Liquid Semisolid Solid (can be liquefied) Solid (cannot be liquefied)

•

Chemical composition Defined (synthetic, chemically defined)

•

Complex ( non-synthetic, not chemically defined)

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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 8/18/12 MDufilho 9

Figure 6.11 Slant tube containing solid media

Slant Butt

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

microbes – contains complex organic substances such as blood, serum, hemoglobin, or special growth factors required by fastidious 8/18/12 MDufilho 11

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 8/18/12 MDufilho 12

Figure 6.12 An example of the use of a selective medium

Bacterial colonies Fungal colonies pH 7.3

pH 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

Durham tube (inverted tube to trap gas) No fermentation Acid fermentation with gas

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

Clamp Chamber

Figure 6.16 An anaerobic culture system

Airtight lid Palladium pellets to catalyze reaction removing O 2 Envelope containing chemicals to release CO 2 and H 2 Petri plates Methylene blue (anaerobic indicator)

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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 A

gar – extract of beef heart and brain MDufilho 18

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 8/18/12 MDufilho 19

Exercise 1.3 – Media Prep

• Important points – Careful measuring of materials – Careful distribution – Sterilization and verification – Storage 8/18/12 MDufilho 20

Weighing Ingredients

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Mixing the Medium

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Dispensing the Medium

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Autoclaving the Media

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Tubed Media

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Pouring Agar Plates

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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 8/18/12 MDufilho 27