Definition of terms...

MR25

MicroChapter6ppt.11thedition.ppt

Home >Nursing homework help >Definition of terms...

Lectures prepared by Christine L. Case

Chapter 6

Microbial Growth

Lectures prepared by Christine L. Case

Microbial Growth

Increase in number of cells, not cell size
Populations
Colonies

The Requirements for Growth

Physical requirements
Temperature
pH
Osmotic pressure
Chemical requirements
Carbon
Nitrogen, sulfur, and phosphorous
Trace elements
Oxygen
Organic growth factor

Physical Requirements

Temperature
Minimum growth temperature
Optimum growth temperature
Maximum growth temperature

Figure 6.1 Typical growth rates of different types of microorganisms in response to temperature.

Psychrophiles

Psychrotrophs

Mesophiles

Thermophiles

Hyperthermophiles

Applications of Microbiology 6.1 A white microbial biofilm is visible on this deep-sea hydrothermal vent. Water is being emitted through the ocean floor at temperatures above 100°C.

Psychrotrophs

Grow between 0°C and 20–30°C
Cause food spoilage

Figure 6.2 Food preservation temperatures.

Temperatures in this range destroy most microbes,

although lower temperatures take more time.

Very slow bacterial growth.

Rapid growth of bacteria; some may produce toxins.

Many bacteria survive; some may grow.

Refrigerator temperatures; may allow slow growth

of spoilage bacteria, very few pathogens.

No significant growth below freezing.

Danger zone

Figure 6.3 The effect of the amount of food on its cooling rate in a refrigerator and its chance of spoilage.

Refrigerator air

5 cm (2′′) deep

15 cm (6′′) deep

Approximate temperature

range at which Bacillus cereus multiplies in rice

Most bacteria grow between pH 6.5 and 7.5
Molds and yeasts grow between pH 5 and 6
Acidophiles grow in acidic environments

Osmotic Pressure

Hypertonic environments, or an increase in salt or sugar, cause plasmolysis
Extreme or obligate halophiles require high osmotic pressure
Facultative halophiles tolerate high osmotic pressure

Figure 6.4 Plasmolysis.

Plasma

membrane

Cell wall

Cytoplasm

H2O

NaCl 10%

Cytoplasm

Plasma

membrane

Cell in isotonic solution.

Plasmolyzed cell in hypertonic solution.

NaCl 0.85%

Chemical Requirements

Carbon
Structural organic molecules, energy source
Chemoheterotrophs use organic carbon sources
Autotrophs use CO2

Chemical Requirements

Nitrogen
In amino acids and proteins
Most bacteria decompose proteins
Some bacteria use NH4+ or NO3–
A few bacteria use N2 in nitrogen fixation

Chemical Requirements

Sulfur
In amino acids, thiamine, and biotin
Most bacteria decompose proteins
Some bacteria use SO42– or H2S
Phosphorus
In DNA, RNA, ATP, and membranes
PO43– is a source of phosphorus

Chemical Requirements

Trace elements
Inorganic elements required in small amounts
Usually as enzyme cofactors

Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria

Singlet oxygen: 1O2− boosted to a higher-energy state
Superoxide free radicals: O2
Peroxide anion: O22–

Hydroxyl radical (OH•)

Toxic Oxygen

Superoxide dismutase

O2 + O2 + 2 H+

H2O2 + O2

Catalase

2 H2O2

2 H2O + O2

Peroxidase

H2O2 + 2 H+

2 H2O

Organic Growth Factors

Organic compounds obtained from the environment
Vitamins, amino acids, purines, and pyrimidines

Biofilms

Microbial communities
Form slime or hydrogels
Bacteria attracted by chemicals via quorum sensing

Figure 6.5 Biofilms.

Clumps of bacteria adhering to surface

Surface

Water currents

Migrating clump of bacteria

Biofilms

Share nutrients
Sheltered from harmful factors

Applications of Microbiology 3.2 Pseudomonas aeruginosa biofilm.

Biofilms

Patients with indwelling catheters received contaminated heparin
Bacterial numbers in contaminated heparin were too low to cause infection
84–421 days after exposure, patients developed infections

Culture Media

Culture medium: nutrients prepared for microbial growth
Sterile: no living microbes
Inoculum: microbes being introduced into medium
Inoculation: the process of introducing microbes into a medium
Culture: microbes growing in/on culture medium

Agar

Complex polysaccharide
Used as solidifying agent for culture media in Petri plates, slants, and deeps
Generally not metabolized by microbes
Liquefies at 100°C
Solidifies at ~40°C

Culture Media

Chemically defined media: exact chemical composition is known
Complex media: extracts and digests of yeasts, meat, or plants
Nutrient broth
Nutrient agar

Table 6.2 A Chemically Defined Medium for Growing a Typical Chemoheterotroph, Such as Escherichia coli

Table 6.3 Defined Culture Medium for Leuconostoc mesenteroides

Table 6.4 Composition of Nutrient Agar, a Complex Medium for the Growth of Heterotrophic Bacteria

Anaerobic Culture Methods

Reducing media
Contain chemicals (thioglycolate or oxyrase) that
combine O2
Heated to drive off O2

Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates.

Lid with

O-ring gasket

Envelope containing sodium bicarbonate and sodium borohydride

Anaerobic indicator (methylene blue)

Petri plates

Clamp with clamp screw

Palladium catalyst pellets

Figure 6.7 An anaerobic chamber.

Arm ports

Air
lock

Capnophiles

Microbes that require high CO2 conditions
CO2 packet
Candle jar

BSL-1: no special precautions
BSL-2: lab coat, gloves, eye protection
BSL-3: biosafety cabinets to prevent airborne transmission
BSL-4: sealed, negative pressure
Exhaust air is filtered twice

Biosafety Levels

Figure 6.8 Technicians in a biosafety level 4 (BSL-4) laboratory.

Make it easy to distinguish colonies of different microbes

Differential Media

Figure 6.9 Blood agar, a differential medium containing red blood cells.

Bacterial colonies

Hemolysis

Suppress unwanted microbes and encourage desired microbes

Selective Media

Figure 6.10 Differential medium.

Uninoculated

Staphylococcus

epidermis

Staphylococcus

aureus

Enrichment Culture

Encourages growth of desired microbe
Assume a soil sample contains a few phenol-degrading bacteria and thousands of
other bacteria
Inoculate phenol-containing culture medium with the soil, and incubate
Transfer 1 ml to another flask of the phenol medium, and incubate
Transfer 1 ml to another flask of the phenol medium, and incubate
Only phenol-metabolizing bacteria will be growing

Obtaining Pure Cultures

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)
The streak plate method is used to isolate pure cultures

Figure 6.11 The streak plate method for isolating pure bacterial cultures.

Colonies

Preserving Bacterial Cultures

Deep-freezing: –50° to –95°C
Lyophilization (freeze-drying): frozen
(–54° to –72°C) and dehydrated in a vacuum

Binary fission
Budding
Conidiospores (actinomycetes)
Fragmentation of filaments

ANIMATION Bacterial Growth: Overview

Reproduction in Prokaryotes

Figure 6.12a Binary fission in bacteria.

Cell elongates and DNA is replicated.

Cell wall and plasma membrane begin to constrict.

Cross-wall forms, completely separating the two DNA copies.

Cells separate.

Cell wall

Plasma membrane

DNA (nucleoid)

(a) A diagram of the sequence of cell division

Figure 6.12b Binary fission in bacteria.

(b) A thin section of a cell of Bacillus licheniformis starting to divide

Cell wall

DNA (nucleoid)

Partially formed cross-wall

Figure 6.13a Cell division.

Figure 6.13b Cell division.

If 100 cells growing for 5 hours produced
1,720,320 cells:

ANIMATION Binary Fission

Generation Time

Number of generations =

Log number of cells (end) − Log number of cells (beginning)

0.301

60 min × hours

Number of generations

Generation time =

= 21 minutes/generation

Figure 6.14 A growth curve for an exponentially increasing population, plotted logarithmically (dashed line) and arithmetically (solid line).

Log10 = 1.51

Log10 = 3.01

Log10 = 4.52

Log10 = 6.02

(1,048,576)

Generations

Log10 of number of cells

Number of cells

(32)

(1024)

(32,768)

(65,536)

(131,072)

(262,144)

(524,288)

Lag Phase

Intense activity preparing for population growth, but no increase in population.

Log Phase

Logarithmic, or exponential, increase in population.

Stationary Phase

Period of equilibrium; microbial deaths balance production of new cells.

Death Phase

Population Is decreasing at a logarithmic rate.

The logarithmic growth in the log phase is due to reproduction by binary fission (bacteria) or mitosis (yeast).

Figure 6.15 Understanding the Bacterial Growth Curve.

Staphylococcus spp.

Figure 6.16 Serial dilutions and plate counts.

Original inoculum

1 ml

9 m broth

in each tube

Dilutions

1:10

1:100

1:1000

1:10,000

1:100,000

1 ml

1:10

1:100

1:1000

1:10,000

1:100,000

(10-1)

(10-2)

(10-3)

(10-4)

(10-5)

Plating

Calculation: Number of colonies on plate × reciprocal of dilution of sample = number of bacteria/ml

(For example, if 54 colonies are on a plate of 1:1000 dilution, then the count is 54 × 1000 = 54,000 bacteria/ml in sample.)

After incubation, count colonies on plates that have
25–250 colonies (CFUs)

Plate Counts

Figure 6.17 Methods of preparing plates for plate counts.

The pour plate method

The spread plate method

Inoculate empty plate.

Add melted nutrient agar.

Swirl to mix.

Colonies grow on and in solidified medium.

1.0 or 0.1 ml

0.1 ml

Bacterial dilution

Inoculate plate containing solid medium.

Spread inoculum over surface evenly.

Colonies grow only on surface of medium.

Figure 6.18 Counting bacteria by filtration.

Multiple tube MPN test
Count positive tubes

Most Probable Number

Figure 6.19a The most probable number (MPN) method.

Volume of Inoculum for Each Set of Five Tubes

(a) Most probable number (MPN) dilution series.

Compare with a statistical table

Most Probable Number

Figure 6.19b The most probable number (MPN) method.

(b) MPN table.

Figure 6.20 Direct microscopic count of bacteria with a Petroff-Hausser cell counter.

Grid with 25 large squares

Cover glass

Slide

Bacterial suspension is added here and fills the shallow volume over the squares by capillary action.

Bacterial suspension

Cover glass

Slide

Cross section of a cell counter.

The depth under the cover glass and the area of the squares are known, so the volume of the bacterial suspension over the squares can be calculated (depth × area).

Microscopic count: All cells in several large squares are

counted, and the numbers are averaged. The large square shown here has 14 bacterial cells.

The volume of fluid over the

large square is 1/1,250,000

of a milliliter. If it contains 14 cells, as shown here, then

there are 14 × 1,250,000 = 17,500,000 cells in a milliliter.

Location of squares

Direct Microscopic Count

Number of bacteria/ml =

Number of cells counted

Volume of area counted

8 × 10−7

= 17,500,000

Figure 6.21 Turbidity estimation of bacterial numbers.

Light source

Light

Blank

Spectrophotometer

Light-sensitive detector

Scattered light that does not reach detector

Bacterial suspension

Measuring Microbial Growth

Direct Methods

Plate counts
Filtration
MPN
Direct microscopic count

Indirect Methods

Turbidity
Metabolic activity
Dry weight