Microbial Growth Physical Requirements of Microbes Temperature (optimal enzyme operation)...

Microbial Growth• Physical Requirements of Microbes

• Temperature (optimal enzyme operation)

• Psychrophiles, mesophiles, thermophiles

• pH (optimal enzyme operation)

• Using buffers in media

• Molds & yeasts versus bacteria

• Chemical Requirements• Carbon source in medium

• Nitrogen, sulfur, phosphorous, trace elements

• Oxygen requirements

• Obligate aerobes, anaerobes, facultative anaerobes

• Free radical oxygen (O2-) and H2O2 dangers; superoxide dismutase and catalase = aerobes

• Culture Media for Microbes• Chemically defined vs. complex media

• Anaerobes: reducing media/Brewer jar

• Other: animals, eggs, tissue culture, CO2

• Media types

• Selective, Differential, Enrichment

• Bacterial Population Growth• Growth Curve: Lag, Log, Stationary, Death

• Quantifying Growth

Characterizing Microbes By Optimal Growth Temperature

Figure 6.1

Temperature Growth Ranges and Food Safety

Figure 6.2

“2-40-140”If > 2 hrs at 40-140oF, don’t eat it!

Physical Requirements: pH

• Most bacteria grow between pH 6.5 and 7.5

• Molds and yeasts grow between pH 5 and 6

Physical Requirements: Osmotic Pressure

• Hypertonic environments, increase salt or sugar, cause plasmolysis

• Extreme or obligate halophiles require high osmotic pressure

• Facultative halophiles tolerate high osmotic pressure















• Media types




• Carbon

• Structural organic molecules, energy source

• Chemoheterotrophs use organic carbon sources

• Autotrophs use CO2

The Requirements for Growth: Chemical Requirements

• Nitrogen• In amino acids, proteins• Most bacteria decompose proteins

• Some bacteria use NH4+ or NO3

• A few bacteria use N2 in nitrogen fixation

• Sulfur• In amino acids, thiamine, biotin• Most bacteria decompose proteins

• Some bacteria use SO42 or H2S

• Phosphorus • In DNA, RNA, ATP, and membranes

• PO43 is a source of phosphorus

• Trace Elements

• Inorganic elements required in small amounts

• Usually as enzyme cofactors

The Requirements for Growth: Chemical Requirements















• Media types




• Singlet oxygen: O2 boosted to a higher-energy state

• Handling superoxide free radicals: O2

2O2- + 2O2

- + 8H+ 4H2O2

oxygen radicals hydrogen peroxide• Superoxide Dismutase (SODS)

• Handling peroxide anion: O22

2H2O2 2H2O + O2

hydrogen peroxide water oxygen gas

Catalase (Peroxidase)

Catalase Test: Bacteria + H2O2 bubbles

How Toxic Forms of Oxygen Are Handled

Obligate aerobes

Faultative anaerobes

Obligate anaerobes

Aerotolerant anaerobes Microaerophiles

Thyoglycollate binds molecular oxygen, reducing it and removing it: R-SH + O2 R-SO2















• Media types




Culture Media: Chemically Defined or Complex

Table 6.2 & 6.4

Anaerobic and Low O2 Culture Methods

Brewer or anaerobic jar CO2 packet

Candle jar

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 tissue culture with low O2, enriched CO2 incubators















• Media types




• Goal: To chemically (or physically) suppress unwanted microbes and encourage desired microbes.

Selective Media

Figure 6.9b, c

Mannitol salt agar : selective for halophiles with 7% salt (osmotic challenge) and differential for mannitol fermenters: good for skin bacterial cultures.

EMB Agar: kills gram positives with eosin and methylene blue, selective for gram negatives. Differential for lactose fermenters. Good for growing enterics.

McConkey Agar: supresses gram positives with crystal violet and bile salts; also differential for

MSA

EMB

MA

• Distinguish between different species based on a metabolic ability.

Differential Media

Figure 6.9a

Blood agar(sheep’s blood) reveals if hemolytic

Mannitol salt agar contains the pH sensitive dye phenol red (yellow when acidic)

Se Sa

• Encourages growth of desired microbe by providing special growth conditions or added growth factors

Enrichment Media

Thioglycollate

Anaerobic or Brewer Jar

Glucose Salts Agar (enriches for microbes that can growth

only on glucose and some inorganic nutrients

Lysed red blood cells provide unique nutrients in

blood/chocolate agar

• A pure culture contains only one species or strain

• A colony is a population of cells arising from a single cell or spore or from a group of attached cells

• A colony is often called a colony-forming unit (CFU)

Pure Cultures Used To Study Characteristics Of A Particular Species















• Media types




Figure 6.12b

Bacterial Growth is Exponential (Logarithmic)

Bacterial “growth” means an increase in the number of individuals, not an increase in cell size.

Figure 6.14

Growth Curve for Bacteria (Logarithmic Plot)

Estimating Bacterial Numbers by Indirect methods

• Direct Measures

• Plate counts of viable bacterial forming colonies

• Counting low viable bacterial numbers by filtration

• Counting viable bacteria with Most Probable Number

• Counting bacteria per ml in direct microscopy

• Indirect Measures

• Turbidity/Absorbance with a spectrophotometer

• Metabolic activity tracking conversion of colored molecules

• Dry weight by weighing a set volume and knowing weight of one cell

• After incubation, count colonies on plates that have 30-300 colonies (CFUs)

Plate Assays: Spread Plate or Pour Plate Methods

Figure 6.15

The dilution in a particular tube = ml of fluid added to tube/total volume after addition; e.g. 1ml/(9ml + 1ml) = 1/10 = 10-2

Direct Measurements of Microbial Growth

Figure 6.19

• Filtration: Good for measuring very dilute samples of bacteria


Figure 6.17a, b

• Multiple tube MPN test

• Count positive tubes and compare to statistical MPN table

• Produces a range of concentrations


Figure 6.18b

Estimating Bacterial Numbers by Indirect methods

• Direct Measures

• Plate counts of viable bacterial forming colonies

• Counting low viable bacterial numbers by filtration

• Counting viable bacteria with Most Probable Number

• Counting bacteria per ml in direct microscopy

• Indirect Measures

• Turbidity/Absorbance with a spectrophotometer

• Metabolic activity tracking conversion of colored molecules/enyzme assay

• Dry weight by weighing a set volume and knowing weight of one cell

• Turbidity

Estimating Bacterial Numbers by Indirect Methods

Figure 620

Metabolic Conversion/Enzyme Assay

• 1 bacterium produces 4.6 x 1012 NADH/sec/cell under idea growth conditions.

• In a 1 ml sample of growing cells, 5.2 x 1023 NADH/sec/ml are produced per second (as revealed by a color-based assay of NADH on the sample)

• Therefore, (4.6 x 1012 NADH/sec/ml) x (5.2 x 1023 NADH/sec/cell) = 2.3 x 1024 cells/ml

Determining dry mass of a fixed volume

An E. coli cell has a dry mass of about 7.0 x 10-19 mg.

A 1 ml sample with a dry mass of 2 mg therefore has:

2 mg/ml x 1 cell/7 x 10-19 mg

= 2.8 x 1020 cells/ml















• Media types




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Microbial Growth Physical Requirements of Microbes Temperature (optimal enzyme operation)...

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