Microbiology Lecture 5 Culturing Microbes PDF

Preview

Summary

Microbiology with Prof. Kucknoor...

Description

I.

II.

Culturing Microbes (5 I’s) A. Inoculation o Inoculation: the introduction of a sample into a container of media to produce a culture of observable growth B. Incubation o Under conditions that allow growth C. Isolation o Separating one species from another o Isolation techniques include:  Streak plate technique  Pour plate technique  Spread plate D. Inspection o Inspection of growth characteristics E. Identification Three Categories of Media A. Three Physical States: 1. Liquid  Broth; does not solidify; usually contains beef extract and peptone 2. Semisolid  Contains solidifying agent (usually Agar)  Agar o Agar: A complex polysaccharide isolated from red algae o Solid at room temp. liquifies at boiling, and does not re-solidify until it cools to 42F o Provides framework to hold moisture and nutrients o Not digestible for most microbes 3. Solid  Firm surface for colony formation  Liquefiable and nonliquefiable  Usually contains beef extract, peptone, and agar B. Chemical Composition: a. Synthetic (chemically defined)  Synthetic: contains pure organic and inorganic compounds in an exact chemical formula b. Nonsynthetic or complex  Complex: contains at least one ingredient that is not chemically definable C. Functional Type 1. General purpose  Grows a broad range of microbes, usually nonsynthetic

III.

IV.

V.

2. Enriched  Contains complex organic substance such as blood, serum, hemoglobin, or special growth factors required by fastidious microbes 3. Selective  Contains one or more agents that inhibit growth of some microbes and encourage growth of others 4. Differential  Allows growth of several types of microbes and displays visible differences among those microbes 5. Anaerobic 6. Transport 7. Assay  Spread plate or pour plate 8. Enumeration Miscellaneous Media A. Reducing Medium o Contains a substance that absorbs oxygen or slows penetration of oxygen into medium; used for growing anaerobic bacteria B. Carbohydrate fermentation medium o Contains sugars that can be fermented, converted to acids, and a pH indication to show this reaction Selective Media  To chemically suppress unwanted microbes and encourage desired a. Mannitol Salt Agar o Selective for halophiles with 7% salt and differential for mannitol fermenters o Good for skin bacterial cultures b. EMB Agar o Kills gram positives with eosin and methylene blue, selective for gram negatives and differential for lactose fermenters o Good for growing enterics c. McConkey Agar o Suppresses gram positives with crystal violet and bile salts; also, differential for enterics Differential Media  Distinguish between different species based on a metabolic ability a. Blood Agar o Sheep’s blood reveals if hemolytic b. Mannitol salt agar o Contains the pH sensitive dye phenol red (yellow when acidic)

VI.

VII.

VIII.

IX.

X.

XI.

Enrichment Media  Encourages growth of desired microbe by providing special growth conditions or added growth factors Unusual Culture Methods  Grows only in certain cell types: Using armadillos to culture M. leprae  Grows only inside live cells: Eggs as culture vessels for influenza virus  Grows only in certain cell types: Using culture with low O2, enriched CO2 incubators Temperature Optima  Each has a minimum, optimum, and maximum growth temperature A. Psychrophiles: cold-loving (-10 to 20C) B. Psychrotrophs (0 to 30C) C. Mesophiles: moderate temperature-loving (10 to 50C) D. Thermophiles: heat loving (40 to 70C) E. Extreme Thermophiles (65 to 110C) pH  Most bacteria grow at 6.5 to 7.5 pH  Acidophiles: 1.0 to 5.5 pH (acidic)  Alkalophiles: 8.5 to 11.5 pH (basic) Osmotic Pressure  Bacteria are better adapted to low OP (i.e tap water)  Plasmolysis occurs at high OP  Solutes (i.e. sugar and salt) limit water availability  Hypertonic environments, increase in salt or sugar, causes plasmolysis  Extreme or obligate halophiles require high osmotic pressure  Facultative halophiles tolerate high osmotic pressure Oxygen  Electron acceptor during aerobic respiration  More efficient than other acceptors (NO3, SO4, and CO3)  Highly reactive oxygen radicals o Singlet oxygen o Superoxide free radicals  Superoxide Dismutase (SOD)  Catalase  Oxygen Requirements: o Obligate aerobes- require O2 o Facultative anaerobes- can use O2 but also grow without it o Obligate anaerobes- die in the presence of O2 o Aerotolerant Anaerobes- do not use O2 but tolerates it o Microaerophiles- requires low amount of oxygen  Anaerobic and Low O2 Culture Methods:

XII.

XIII.

XIV.

XV.

A. Candle Jar B. CO2 Packet C. Brewer or Anaerobic jar Bacterial Division  Bacteria divide by binary fission  Alternative means: o Budding o Conidiospores (filamentous bacteria) o Fragmentation Generation Time  Generation time: time required for cell to divide/ for population to double  Average for bacteria is 1-3 hours  E. coli generation time is 20 min o 20 generation (7 hrs), 1 cell becomes 1 million Bacterial Growth  Bacterial “growth” means an increase in the number of individuals, not an increase in cell size  Phases: 1. Lag Phase o Making new enzymes in response to new medium 2. Log Phase o Exponential growth o Desired for maximum yield of products o Most sensitive to drugs and radiation during this period 3. Stationary Phase o Nutrients become limiting or waste products becoming toxic o Death rate= division rate 4. Death Phase o Death exceed division Estimating Bacterial Numbers A. Direct Measures: to count individual cells per ml in direct microscopy o Plate counts of viable bacterial forming colonies o Counting low viable bacterial numbers by filtration, good for measuring very dilute samples of bacteria o Counting viable bacteria with most probable number  Counts positive tubes and compares them to statistical MPN table  Produces a range of concentrations B. Indirect Measures: measures the effects of cell growth o Tubidity/Absorbance with a spectrophotometer

o Metabolic Activity tracking conversion of colored molecules o Dry weight by weighing a set volume and knowing weight of one cell...