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

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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 28/18/12 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

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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 48/18/12 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

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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?

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Categories of Media Physical States • Liquid

• Semisolid

• Solid (can be liquefied)

• Solid (cannot be liquefied)

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Chemical composition• Defined (synthetic, chemically defined)

• Complex ( non-synthetic, not chemically defined)

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Physical States of MediaLiquid – broth; does not solidify

Semisolid – contains solidifying agent

Solid – firm surface for colony formation

– Contains solidifying agent

– Liquefiable and nonliquefiable

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Figure 6.11 Slant tube containing solid media

Slant

Butt

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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

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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

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Figure 6.12 An example of the use of a selective medium

Fungal coloniesBacterial 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

No fermentation Acid fermentationwith gas

Durham tube(inverted tubeto 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

Envelopecontainingchemicals torelease CO2

and H2

Palladium pelletsto catalyze reactionremoving O2

Methylene blue(anaerobicindicator)

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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 Agar – extract of beef heart and brain

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Agar

Most commonly used solidifying agent is agar

• A complex polysaccharide isolated from red algae– Solid at room temperature, liquefies at boiling

(100oC), does not re-solidify until it cools to 42oC

– Provides framework to hold moisture and nutrients

– Not digestible for most microbes

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Exercise 1.3 – Media Prep • Important points

– Careful measuring of materials– Careful distribution– Sterilization and verification– Storage

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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 – 25o and 37o

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