microbio--MODULE 3. BACTERIAL GROWTH

Explain bacterial reproduction via binary fission. (4 steps)

It is an asexual process. Relatively simple compared to eukaryotes. Offspring are genetically identical and start with one set of DNA, and end with two. 

• Cell elongation (due to intake of nutrients from environment and their conversion into new cellular material) and chromosomal replication (double it and occurs simultaneously with cell elongation)

• Cell wall invagination as cell envelopes begin to grow inward between nuclear bodies

• Transverse cell wall formation where the cross wall forms between the two cells

• Then the two cells separate (offspring are genetically identical)

Define and distinguish between bacterial growth and generation time.

Bacterial growth: increase in total number of bacteria, refers to changes in a whole population of bacterial cells than changes to a single organism gaining size

Generation time: “doubling time” is the time interval required for the bacterial cell population to double. The prevailing means of bacterial reproduction is binary fission where one cell divides into two identical cells. Thus, with a single bacterium, the increase in population is a geometric progression (1-2-4-8-16-32-64, etc)

• “how long it takes for one cycle to occur where one cell becomes two”

Diagram a typical bacterial growth curve: identify and differentiate the 4 phases.

can have different growth rates, but all have the same phases (lag, log, stationary, death).

Describe processes occurring during each of the four phases of bacterial growth.

Basically how fast an infection can progress

Lag phase: preparation for binary fission, bacteria prepare to divide by gathering nutrients, synthesizing new protoplasm, elongating cell, and replicating chromosomes (looks like nothing is going on)

Exponential (log) phase: bacterial numbers increase logarthymically because of many cell divisions (binary fission has started)

Stationary phase: nutrients become limiting and bacterial number remains constant (environment can’t support more growth)

Death phase: number of nonviable bacteria exceeds number of viable cells

List factors that affect generation time of bacterial reproduction. Cite and explain examples of each. (3)

Different species have different generation times. 

• Ex: TB has 24 hr cycle vs 20 min for e. coli

differences in nutrients available in environment (milk vs broth) 

• Ex: broth is 48 min and milk is 26

physical conditions (pH, temp, humidity, gaseous composition, light, pressure, etc)

desiccation

drying that is detrimental to bacterial growth

phototroph:

E source is light via photosynthesis

chemotroph

E source is chemical

autotroph

C source is CO2–microbe builds up organic compounds

heterotroph

C source is organic–microbe breaks down organic compounds (like us)

thermophile

likes it hot (46-65 deg C or 115-149 deg F)

mesophile

 like moderate temps in 20-25 deg C range with optimum at 37 deg C (69-113; 98.6 deg F)

psychrophile

like it cool to cold (less than 20 deg C or 68 deg F)

aerobe

grow in presence of free atm O2

anaerobe

cannot grow in presence of free atm O2

facultative anaerobe (also known as facultative):

can grow in O2 rich or absent environments bc possess enzymes for both metabolic pathways

capnophile:

req 3-10% inc in CO2 conc to initiate growth

halophile

require high salt conc for life (ex: bacteria flora in sea)

Name the three major nutritional requirements and five physical factors affecting bacterial growth (dive deep into nutritional needs (3), temp, pH, and gas comp.). Explain their importance, effect and optimal conditions for growth.

Need nutrition requirements met and certain physical conditions to be known for each organism to identify agents causing infection in a clinical setting. (prerequisite for the study of microbes in the lab is the ability to grow the bacteria)

Nutritional needs: 

• Main–water (major component of the cytoplasm bc 75-80% and drying/dessication is detrimental to bacterial growth), energy source (phototroph where the energy source is light via photosynthesis or chemotroph where energy source is chemical), and a carbon source (autotroph where C source is CO2–microbe builds up organic compounds or heterotroph where C source is organic like glucose–microbe breaks down organic compounds)

• Main needs are like ours

• others–vitamins (catalysts that increase reaction speeds), amino acids, purine, pyrimidines, cations: chemical transporters and enzyme co-factors (Na+, K+, Ca2+, Mg2+)

Physical conditions: environmental factors influence the growth rate and must be considered when bacteria are cultured in a lab–temp, pH, gas composition, light, pressure

• Temp: determines rate of chemical reactions and metabolism, rate of growth and total amount of growth, and microbe morphology (shape)

  • For each species, there is a minimum, optimum, and maximum temp that allows growth. Different microbes have different temp optimums (where grow best)

  • Psychrophiles like it cool to cold (less than 20 deg C or 68 deg F), mesophiles like moderate temps from 20-45 deg C or 68-113 deg F *optimum at 37 deg C or 98.6 deg F–which is body temp (room temp is 20-23 deg C or 68-73 deg F), and thermophiles like it hot from 46-65 deg C or 115-149 deg F

• pH or hydrogen ion concentration: microbes grow best at specific pH

  • Optimum pH: 6.5-7.5

  • Extreme pH: 4-6.5 and 7.5-9

  • Most human pathogens grow best in neutral to slightly alkaline (7.25-7.45) environments

• Gas composition: the main gases that affect bacterial growth are O2 and CO2. bacteria display a wide variety of responses to free O2 and are divided into: 

  • Aerobes: grow in presence of free atmospheric O2

  • Anaerobes: cannot grow in presence of free O2

    • Obligate anaerobes have to live in anaerobic air, facultative anaerobes can live in both because have metabolism pathways for both air environments, and aerotolerant can tolerate slightly aerobic conditions

  • Capnophiles require 3-10% increase in CO2 concentration to initiate growth

Discuss classifications of bacteria determined by their response to oxygen (aerobe, anaerobe, etc), distinguish one classification from another and recognize the type of oxygen response when given a photo or description of growth characteristics of a bacterium.

 Obligate aerobes–require O2, grow at top of tube where more free O2

Facultative anaerobes–grow with or without O2, grow throughout since doesn’t matter

Obligate anaerobes–free O2 is toxic, grow at bottom of tube with least O2

Aerotolerant anaerobes–tolerate free O2, grow throughout

Microaerophiles–prefer some free O2, grow in the middle

Thioglycollate media is used to demonstrate this

Given a specific range of light, identify the general effect of that wave length on bacterial growth.

trend: the smaller the wavelength, the more likely to kill the bacteria. Direct sunlight can help or hurt, but depends on desiccation/presence of water.

State the effect of pressure on bacteria. Identify the most common causative agents.

• which bacteria is salt tolerant /halophilic

Bacterial cells are encased in a semipermeable membrane (allows H2O to pass in/out but offers resistance to dissolved substances). Must be aware of pressure so must regulate what it lets in. Most bacteria resist small changes in osmotic pressure (from letting in sugar or salt ions and water following either filling up or shrinking the cell) but are killed or inhibited by large changes, usually due to high concentrations of salt or sugar.

• Ex: in food industry we use salt and sugar preservation to keep bacteria from growing because of its high concentration

• Some bacteria require high salt concentration for like (halophilic bacteria like the bacteria flora in the sea)

• Most bacteria do not tolerate high amounts of salt

• Some organisms are very salt tolerant (staphylococcus tolerates 7.5% which is twice salt concentration in the sea, and this is useful for identification purposes by making a media specific with high salt that only staph can grow in)

State the requirements necessary to successfully grow bacteria in a lab setting being sure to address a sterile environment. Dive into what media is.

2 basic components of media

To grow bacteria successfully in the lab, one must simulate the natural habitat of the bacteria (via nutrition, temp, pH, gas composition, light and pressure). Culture media must be sterile (what grows is sample not contaminants). 

Media: 

• Media can be liquid (broth), solid (due to agar concentration of 1-2%), or semi-solid (due to agar concentration of .05-.5%)--an example of this last one is a motility medium of .5% agar to ID motility of bacteria

• Basic components of media is an energy source (sugars especially glucose) and protein or meat based additives that are rich in C and N like trypticase soy broth or agar

• Media is rich in nutrients and one can add to it to inc/dec growth of bacteria

enriched:

media containing additional growth factors beyond that of normal media to enhance growth of all bacteria, especially fastidious (picky) ones. 

Ex of enriched media and growth factors: blood–blood agar plates or BAP, or chocolate–contains additional nutrients in the readily accessible form of boiled blood so don’t have to expend energy to digest components

enrichment

 used in clinical micro labs for intestinal specimens to increase likelihood of isolating intestinal pathogens

- one particular type of media specific for intestinal samples

- important since there are many normal bacteria in GI tract

- generally a broth media

- enhances growth of pathogens and inhibits growth of normal flora

- not always needed if high enough levels of pathogen, but useful to ID in samples

selective (example of dyes as indicator/inhibitor and antibiotics)

• what bac type inhibitors usually inhibit against

• CNA agar selective against what, composition

• ex dyes used as indicators or inhibitors:

promotes growth of one organism and slows growth of others (using dyes, high salt concentrations, antibiotics). 

- Some selects for growth; some selects against growth of others

1. Indicator:

   1. Phenol red: red when alkaline and yellow when acidic

2. Inhibitor: inhibit growth of specific bacteria—like gram pos bacteria (especially gram positive cocci)

   1. Gentian violet, eosin Y, methylene blue

   2. Allows for growth of gram negative bacteria

• Ex columbia agar with antibiotics (columbia CNA agar):

  • Selective for gram pos organisms (esp cocci)

  • Addition of antibiotics colistin and nalidixic acid (“CNA”) 

  • Causes inhibition of gram neg growth thereby allowing for growth of gram positive organisms

differential (SBA hemolysis 3 result meanings)

allows for the distinguishing (differentiation) of organisms growing on plated media due to a substance included in the media

- ex: colony selected to grow pink and everything else yellow

- set up diff culture plates depending on the specimen (e.g. diff for throat vs intestine samples)

- ex: sheep blood agar (SBA):

- does not select for growth of any specific organism but does differentiate organisms on the basis of hemolysis

- bacteria will grow bc nutrition: beta hemolysis (total clearing bc enzymes that digest RBC totally digest all RBC), alpha hemolysis (greening where bacteria have ability to do partial digestion of RBC), and gamma hemolysis (no hemolytic reaction and instead simply grows on media)-->used to ID strep which portrays beta

selective versus differential

select (selective) for growth of specific organism and of those that grow, can be made more distinct by differentiating between diff organisms (differential)

enriched versus enrichment

enriched is general media with added nutrients, enrichment is one particular type of media specifically used for intestinal samples

Discuss one application of the use of selective and differential media (MacConkey Agar and Mannitol salt agar)

differential vs selective agents of each agar

MacConkey Agar

• Lactose fermenting/nonfermenting GNR

• Inhibits gram pos bacteria thereby selecting for growth of gram neg (selective)

• Differentiates lactose fermenting GNR (+) from lactose nonfermenting GNR (-) (differential)

• Lactose neg can’t use lactose for nutrition

• On normal blood agar, would not see the distinction between 2 organisms and would think there is only one present

• Lactose fermenting/nonfermenting Klebsiella pneumoniae and shigella sonnei

  • Selective bc inhibits most gran pos bacteria due to presence of bile salts and crystal violet thereby selecting for growth of gram neg 

  • Differential bc distinguishes on the basis of lactose fermentation: fermenters are pink and nonfermenters are clear

  • Has mucoid appearance bc of capsule

• differential agent=lactose, selective agent=bile salts (gram neg only can grow)

Mannitol salt agar

• Selective bc inhibits most bacterial growth (gram pos and neg) due to high salt conc (7.5%) in medium

• Differential on basis of fermentation of mannitol (sole sugar in formulation)

  • Fermenters are yellow colonies–acidic end product of metabolism– (s. aureus) and nonfermenters are red/pink (other staph or micrococcus)

  • Same genus, diff species

• Differential agent=mannitol and selective agent=salt

Describe steps involved in handling patient samples in the clinical microbiology laboratory. (10)

• Specimen collection, id of specimen-appropriate media, set up cultures, streaking for isolation (look in center where most diluted and can see indiv colonies), incubation of cultures (37 for mesophiles), reading of culture plates to determine biochemical tests needed for bacterial ID, biochemical testing for bacterial ID (with coagulate tubes, test strips, catalase–O2 released as gas after HCl added–, or nutrient reactions), and antimicrobic susceptibility testing (what antibiotics organisms are susceptible/resistant to)