lab 1 safety and lab guidelines syllabus expectations –12 quizzes – drop 1 – no make up...
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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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