BIOL 251 CH 9 Study Points

Pure culture

A population of microorganisms descended from a single cell

Mixed culture

More than one species growing together

Define and explain what a colony is

group of a single microorganism

• single colony is a pure culture

Explain binary fission

bacterial reproduction, asexual

1. cell gets longer, DNA replicates

2. DNA moved into future daughter cell, cross wall forms

3. cell divides into two cells

4. cells separate

Describe exponential growth

growth that scales with size, bigger the population the faster it grows

• usually expressed 2^n

growth curve

1. lag phase: no increase

2. log phase: exponential growth

3. stationary phase: plateau in living cells, rate of cell division and death equal

4. death/decline phase: exponential decrease

What kind of information can be derived from a growth curve?

• responses to environment/stimuli

• doubling time

• carrying capacity

serial dilution

dilution of the original culture by multiples of 10 to obtain plates with CFUs between 30-300

Counting via hemocytometer

counts red blood cells

• easy to use, relatively fast, and inexpensive

• the counting chamber does not work well with dilute cultures because there may not be enough cells to count

Counting cells via Coulter Counter

counts individual cells as it passes thru the detector

• rapid and accurate within a range of concentrations

• however, if the culture is too concentrated, more than one cell may pass through the aperture at any given time and skew the results.

counting by measuring Turbidity

indirect cell count, uses a spectrophotometer to measure the turbidity of a sample

• more turbid sample contains more microbial cells

• fast method to estimate cell density as long as there are enough cells in a sample

counting using Membrane Filtration

directly count the number of microbial cells present

• works if the sample is more dilute and in a larger volume

Explain what a direct plate count is and how it is done

used to calculate living bacteria in sample

1. serial dilution

2. plating sample

   1. spread plate

   2. pour plate

3. incubation

4. counting colonies

   1. 30-300 CFU range

5. CFU formula calculation

Obligate aerobes

requires oxygen

• grows at the top of tube

Obligate anaerobes

grow in the absence of oxygen (intolerant of oxygen)

• grows at bottom of tube

Facultative anaerobes

grow in aerobic or anaerobic conditions, thrives more in oxygen

• heavy growth at the top of the tube and growth throughout the tube

Aerotolerant anaerobes

(anaerobic respiration) indifferent to the presence of oxygen

• equal growth throughout

Microaerophiles

require between low atmospheric oxygen levels (5 – 15%)

• grows slightly below the top

Explain how microbes tolerate ROS

enzymes break down ROS, electron donor (reduced compound) oxidizes peroxides to water

• superoxide dismutase

• peroxidase

• catalase

What is the catalase test used for? Explain how a positive result is achieved

detects presence of catalase enzyme

• positive result shown by formation of bubbles

Know about the C. perfringens example

C. perfringens is an obligate anaerobe meaning it grows that grows devoid of oxygen

• dead tissue no longer supplied by oxygen makes an ideal environment for its growth

• dead tissue will have to be removed (debrided)

pH and bacteria

measure of H+ ion concentration

• Microbes have an optimum, minimum and max

• Highly acidic or alkaline environments inhibit bacterial growth

types of bacteria:

• Neutrophiles: pH range is 5–8

• Acidophiles: Optimal pH is below 5.5

• Alkaliphiles: Optimal pH above 8.5

pH food preservation

Lemon juice, vinegar, and pickled foods are used in preservation as highly acidic or alkaline environments inhibit bacterial growth

• pH of vinegar is about 2.5 - 3.0

H. pylori example

this bacteria is able to survive the extreme acidity of the stomach despite being a neutrophile

how?

• creates a microenvironment in which the pH is nearly neutral

• produces large amounts of the enzyme urease, which breaks down urea to form NH4 + and CO2, ammonium ion raises the pH of the

testing?

• patient given urea containing radioactively labeled carbon atoms

• if H. pylori present, it will break down urea producing radioactive CO2 in patient’s breath

• uses flagella to corkscrew through mucosal lining, mucus production decreases, stomach lining not protected from acidic environment

• Cause of most peptic ulcers

temperature effect on microbes

the optimum temperature (highest growth rate temp.) depends on the species

practical use:

• Food preservation

  • refrigeration prevents spoilage

  • freezing temps microbes unable to grow and divide

• Infectious diseases

  • microbes able to grow at body temp, 37 C

temperature range types

• psychrophile: -5 to 15 C

• mesophile: 20 to 30 C

• thermophile: 45 to C 70

• hyperthermophile: 70 C or greater

L. monocytogenes example

• Ubiquitous in environment

• known to survive under various conditions of refrigeration, freezing, heating, and drying

• Transmission via contaminated foods; problem for food industries

  • Can form biofilms on food processing equipment

• foods that do not require further cooking are under great risk

• Immunocompromised and pregnant are most susceptible

  • problematic for fetus (can cause placental barrier)

Understand solute (salt) concentration and food preservation

High salt concentrations are used to preserved foods, causes plasmolysis

• Foods with high dissolved solute (salt or sugar) are naturally preserved

  • Jams, honey

  • Mustard, ketchup

Know about the S. aureus example

S. aureus can survive in high salt concentrations

• Associated with improper food handling and warm storage

• Causes rapid-onset gastroenteritis without bacteria in the bloodstream

Understand/explain how bacteria are cultured in the laboratory

• Must know growth and nutritional requirements

• Environmental requirements must be known

• Broth cultures or agar plates

selective vs differential media

• selective - allow some organisms to grow while inhibiting others

• differential - Distinguish between organisms that all grow

Blood agar plates

Contain nutrient media and 5% sheep red blood cells

• Routine clinical use to grow hemolytic bacteria

• An example of complex and differential media

MacConkey agar plates

Selects for gram negative bacteria

• An example of selective and differential media

  • Lactose makes it differential

nutrient agar

used for routine lab work, general purpose, supports growth of a variety of nonfastidious bacteria

• not selective or differential

Mannitol Salt Agar (MSA)

• Selective: high salt

• Differential: mannitol fermentation

Thayer-Martin agar

• Selective (antibiotics) - selects for (fastidious pathogens)

• Used for Neisseria

Chocolate agar

• Not selective or differential

• Used for fastidious bacteria - picky organisms

Environmental needs of bacteria

Incubators

• CO2 and Non-CO2 incubators

• Stationary and shaking incubators