CHAPTER SUMMARY OF UNIT 2
Chapter 6 – Bacteria Growth
I. GROWTH AND CELL DIVISION
1. Microbial growth defined:

Microbial growth can be defined as orderly increase in quantity of all cell components
and in number of cells of an organism.

Because of limited increase in cell size and the frequency of cell division seen in bacteria,
growth is measured in cell number.

The fasion of cell division in most bacteria is binary fission, in which the chromosome
makes exact copy and the cell forms a transverse septum that separate the original cell
into two identical cells. Bacteria do not undergo a cell cycle as in mitosis.

Yeast cells and some bacteria divide by budding, in which a small new cell develops
from the surface of an existing cell.
2. Phases of growth:

In a nutrient rich medium ( a mixture of substances on or in which microorganisms grow,
can be liquid or solid), bacteria divide rapidly. The length of time required for one
division is called the generation time. Such growth is said to occur at an exponential, or
logarithmic, rate.

Bacteria introduced into a fresh, nutrient-rich medium disply non-syncronized growth
within certain range of time. Over time, there are four major phases of growth: (1) In lag
phase, the organisms are metabolically active – growing and synthesizing various
substances but not increasing in number. (2) In the log phase, organisms divide at an
exponential, or logrithmic rate and with a constant generation time. these properties og
growth in the log phase can be used to calculate both the number of generation and the
generation time. (3) In the stationary phase, the numbers of new cells produced equals
the number of cells dying. The medium contains limited amounts of nutrients and may
contain toxic quantities of waste materials. (4) In the decline phase, or death phase,
many cells lose their ability to divide and eventually die. A logrithmic decrease in the
number of cells results.

Growth in colonies paralelles that in liquid medium, exceptthat most growth occurs at the
edge of the colony, and all phases of growth occur simultaneously somewhere in the
colony.
3. Measuring bacterial growth = measuring bacteria concentration at certain time points

Serial dilution: Successive 1:10 dilutions of a liquid culture of bacteria are made and
tranfered onto an agar plate; the colonies that arise are counted. Colony Forming Unit,
CFU = number of colonies from 1 ml of liquid culture x times of dilution.

Direct count: Use a Petroff-Hausser counting chamber (hemocytometer). Can not
determine if living.

Most propable number (MPN)

Filtration

Measuring OD600nm: Use a spectrophotometer to measure light obsorption that represents
turbidity. This is the only method that can monitor the growth in real time and harvest
cells at the log phase, which is necessary in a series of experiments.
II. FACTORS AFFECTING GROWTH
1. Physical factors

All bacteria have optimum of the following.
o temperature
o pH
(Effect of buffer is to reduce change in pH)
o O2 level
o moisture
o nutrients
o salt or pressure

Obligates and trophiles:
o Obligate aerobes: Must have O2; e.g. Pseudomonas
o Obligate anaerobes*: Must have no O2; e.g. Clostridium botulium
o Faculative anaerobics): Grow well with O2 and grow less well without O2 ; e.g.
E. coli, Staphylococcus
o Aerotolerant: Can survive O2 but can not grow in it;
o Microaerophiles: Samll quantity of O2; Grow below surface of agar; e.g.
Campylobactor
o Capnophiles: High CO2, low O2;
o Psychrophiles: Low temperature, <25°C;
o Mesophiles: 25°C to 40°C;
o Thermophiles: > 40°C, best 50-60°C, even seen at 110°C;
o Halophiles: High salt; Can live in sea water (3% salt) and grow well in 7.5% salt.
o Barophiles: High pressure, even 7000m under the sea;
o Acidophile: pH 0.1 – 5.4; e.g. Lactobacillus
o Neutrophile: pH 5.4 – 8.0; Most bacteria here;
o Alkaliphile: pH 7.0 – 11.5; e.g. Vibrio cholera, Argrobacterium
* O2 does not kill the obligate anaerobes but superoxide O2- does. The following reactions are
catalyzed by superoxide dismutase and catalase, whch are missing in obligate anaerobes.
Obligate aerobes and faculative anaerobes have both; aerotolerants misses the catalase.
Superoxide ------- O2 + H2O2
H2O2--------CO2 + H2O
2. Nutritional factors:

All organisms require a carbon source: (1) Autotrophs use CO2 in the air to synthesize
other substances. (2) Heterotrophs require glucose or other organic carbon source from
which they obtain energy and intermediates for synthetic processes. A typical medium
contains peptone, yeast extract, glucose, salt.

Microorganisms require an organic or inorganic nitrogen source from which to
synthesize protein and nucleic acid.

Others: S, P, K, Fe, vitamins, and trace elements.

Nutritional complexity reflects a defficiency in biosynthetic enzymes.

Microorganisms adjust to limited nutritional supplies by increasing the quantities of
enzymes they produced, by making enzymes to metabolize another available nutrient. Or
by adjusting their metabolic activities to grow at a rate consistent with available nutrients.

Fastidious bacteria: Have special nutritional need, picky eaters.
III. SPORULATION
It let bacteria withstand long period of drought and high temperature. When favorable condition
restored, germination occur. Endospores begin to develop into vegetative cells.
IV. CULTURING BACTERIA
1. methods of obtaining pure cultures:

Streak plate method

Pour plate method
2. Culture media:

Selective media: Allowing some to grow but not others. E.g. amp+, tet+ plates

Differential media: E.g. MacConkey media has crystal violet, bile salt, lactose and pH
indicator. Only G- can grow. Lactose fermenter = red; non-fermentor = colorless.

Enrichment media: Provide nutrition that foster growth of a particular organism. E.g.
Samonella typhi grow well in the presence of Selenium.

Many kits and devises available, looking for color change, air bubbles etc. to make
diagnosis.
Chapter 7 – Sterilization and Disinfection
I. THE CONTROL OF MICROBIAL GROWTH
1. Terms defined:

Sterilization: Remove or kill all microorganism in or on an object. Sterility: Less than
1/106 chance of finding an organism.

Disinfection: Reducing pathogenic microorganisms in or on an object so that there is no
threat of disease.

Disinfectant: A chemical agent applied on inanimate object to kill microorganisms. Most
disinfectants do not kill endospores.

Antiseptic: A chemical agent that can be applied safely on living tissue. Kill or inhibit
microorganisms.

Sanitizer: A chemical agent that can be used safely on food handling utensils in order to
meet public safety standard.

Bactericide: Kills bacteria. Most such agent do not kill endospores.

Bacteristatic: Inhibit growth of bacteria.

Viricide, fungicide, sporocide: You get it.
2. Because of the logarithmic death rate, a defined proportion of organisms die in a given time
interval. The fewer microbes present, the less time will be needed to achieve sterility.
Microorganisms differ in susceptability so the rates are different.
II. CHEMICAL AGENTS
1. Potency, or effectiveness:

Affected by time, temperature, pH, and concentration of the agent.

Evaluated by phenol coefficient: the ratio of the dilution of the agent to the dilution of
phenol that will kill organisms in 10 minutes but not in 5 minutes.

Filter paper test: Measured in mm.

Dilution test: Dry dip 10 minutes, rinse with water, incubate in 2 ml culture. Measured as
the highest dilution to inhibit growth.
2. Selection of agent: Time, safety, penetration, easy to use, cost, odor etc.
3. Mechanism of action: If disrupt covalent bonds, it sterilize; if disrupt non-covalent bonds, it
can only disinfect.

Protein denaturants:E.g. boiling, weak acid, ethanol, formamide, formaldehyde,
halogens.

Protein degradation: E.g. strong acid, fire.

Membrane surfactant: E.g. detergent.
4. Examples:

Heavy metal: effective on virus

Quaternary amonium (quats, NH4+): Zaphiran, Windex, ear piercing kit

Acids, alkalines

Phenol: Lysol (orthophenylphenol), Amphyl, Hexachlorophene, Dichlorophene.

Oxidizing: H2O2, potassium permanganate

Ethylene oxide gas: penetrating and sterilizing, good for small chamber

Dyes, salt: E.g. pickled food
III. PHYSICAL AGENTS
1. Heat:

Dry heat such as in oven or flame: Flame is sterilizing.

Moist heat: Autoclav is the most commonly used way in labs and hospitals. 121°C,
15bl/in2 for 15 to 20 minutes or till the indicator turns possitive. Kills and destroys
endospore and all spores, biofilm that is resistant to chemical wash, vitus and DNA.

Pasteurization: Kills most pathogens such as Salmonella and Mycobacterium in milk
and beer, but not sterilize. 71.6°C for 15 minutes or 62.9°C for 30 minutes.
2. Other methods:

Freeze and dry such as lyophilization

Drying / reducing moisture

Radiation: UV, sun, irradiation of food, microwave

Ultrasound

Filter: 0.2 m for filter sterilizing solution that is heat sensitive and can not be autoclaved
Chapter 20 – Antimicrobial Drugs
I. CONCEPTS
1. Chemotherapy is the use of any chemical agent in the treatment of disease.
2. Chemotherapeutic agent, or drug, is any chemical agent used in medical practice.
3. Antimicrobial agent is a chemical agent used to treat a disease caused by microbe.
4. Antibiotic is a chemical substance produced by microorganisms that inhibits the growth of or
destroys other microorganisms.
II. GENERAL PROPERTIES OF ANTIBIOTICS
1. Selective toxicity: ED50/LD50
2. The spectrum of activity: Usually there are antibiotics agaist G+ bacteria or broad spectrum
ones agaist both G+ and G-.
3. Modes of action:

Inhibit cell wall synthesis by inhibiting the synthesis of peptidylglycan.

Disrupt the structure or function of cellular membrane.

Antimetabolites

Protein synthesis (translation) inhibitors

Nucleic acid synthesis (replication of DNA or transcription of RNA) inhibitors
4. Common side effects:

Most drugs are metabolized in liver, resulting in liver damage.

Most drugs are eliminated in kidney, resulting in kidney damage.

Some have special toxicity to inner ear, resulting in hearing damage.

Allergy

Disrupt normal microflora, resulting in superinfection.
5. Antibiotic resistance

Results of natural selection for antibiotic resistant mutations or swapping genetic material
containing antibiotic resistant genes with each other

Mechanism: Induce or synthsize -
o Enzyme that can destroy drugs
o Chemical analogue competing entry into the cell
o Memebrane channels or “pumps” for rapidly efflux of drugs
o Altered target sites
o Prevent antibiotic resistance: “overwhelming or none” – Do not use unless
absolutely necessary; but use large enough dosage for long enough time.
6. Measuring effect of anitibiotics

Most common: disc infusion (Kirby-Bauer) method and minimum dilution time
IV. REPRESENTATIVE ANTIBIOTICS
Name
Spectrum
Mechanism
Common Side Effects
Sulfonamide
Broad
Antimetabolite –
Kidney damage
Inhibit synthesis of folic acid
Nausea
(sulfa drugs)
rash
Penicillins
G+
Disrupt cell wall synthesis by
Allergy
blocking the synthesis or cross-
Cephalosporins
linking of the peptidylglycan
Vancomycin
Bacitricin
Polymyxin
G-
Disrupt cellular membrane
Highly toxic
Only used topically
Or monitor kidney
function as in-patient
a.Streptomycin
Broad
Protein synthesis inhibitor
b.Tetracycline
a. Kidney, Ear
b. Teeth
c.Chloramphenical
c. Bone marrow –
d.Erythromycin
aplastic anemia
a.Rifampin
a.TB
a. Transcription
a. Red skin, liver kidney
b.Quinolone
b.Protozoa,
urinary tract
infection
b. Replication
b. Nausea, headache
How to review?
1. All the terms and definitions that are in black.
2. The framework of the above summaries.
3. There is NO such types of questions:
1). Given a serial dilution result, calculate back the CFU/ml of the iriginal culture.
2). Fission, sporing and budding – What is the difference in life cycles. (But need to
know bacteria reproduce by fission that is not mitosis)
3). Calculating MPN according to results
4). Mechanism of aerobic and anaerobic metabolism e.g. lacking of catalase
5). Difference in streak and pour plates
6). Mechanisms of action of chemical disinfectant e.g. protein denaturation
7). Mechanisms of anitibiotic resistance e.g. to induce -lactamase to degrade drugs
4. Will include the following types of questions:
1). Growing curve comparisons of all the obligates, faculatives, and trophiles under
favored and unfavored conditions
2). Dynamics of the growth curve
3). From results, find minimum dilution of effective disinfectants or antibiotics
4). How different oxygen requirements (5 of them) affect growth pattern in solid culre in
test tubes (surface, middle, butt, or under surface).
5). Results of zone inhibition
6). Given culture composition, judge which one can culture autotrophs