Methods in micro cult and phys

What are the 3 domains of life

1)Archaea-extremophiles

2)Bacteria

3)Eukarya -prokaryotes

Ways to differentiate bacteria on phenotypic characteristics?

1) Morphology

2) Culturing conditions

3)Physiology

4)Pathogenic characteristics

Shape is indicative of what?

Function

Gram postive cell structure vrs Gram neg

Gram positive: thin peptidoglycan (polymer that gives cell wall strength cross linking via AA),

Gram neg: thick layer of peptidoglycan, outer and inner membranes

what does mycoplasma mean?

-no cell wall

In gram staining process what will happen w Gram neg and Gram pos

-Gram neg will not be stained and be pink

-Gram positive will be stained purple

Gram staining procedure?

1) add methanol to fix cells to surface

2)add crystal violet

3) add iodine which binds stain to gram positive cells

4) wash w ethanol-removes purple from gram neg

5)add safranin counterstain to show gram neg which will be pink and Gram positive will be purple

What happens to gram pos and gram neg in KOH procedure

-Gram positive cells rigid, add kOH cell stays intact DNA stays inside

-Gram negative, KOH punches holes DNA comes out bc weak peptidoglycan

Unnexpected results in GRam staining?

1) wrong procedure

2)old gram positive bacteria could be stained pink previosly

3) atypical cell wall

Mycobacteria cell wall structure simular to what?

-Gram positive but it has an outer layer of waxy mycolic acid that prevents dye penetration

What gene is in all living things?

-16 rRNA each thing has a unique one

How many mutations every million years?

1

How many variable regions are in 16rRNA gene?

9: variable regions have been used to classify and identify bacteria: we analyze the seq of dna that encodes it not the RNA molecule

Advantages of bacteria identification using 16S rRNA gene sequence?

1) do not need to grow or culture bacteria only need presence of bacterial DNA

2) less time

Disadvantages of bacterial identification using 16S rRNA gene seq?

• dependent on matching to a known sequence 95% same is considered same genus

• lacks resolution

• cannot reveal metabolic capabilities of the bacteria= does not reveal functional diversity

• high risk of contamination

Meaning of species for plants and animals?

-can interbreed to make fertile offspring

does NOT apply to bacteria bc its asexual

What is the most continoulsy living domain?

-cyannobacteria

Antibiotic resistanance partially due to

not taking full course of antibiotics

Factors to consider when trying to grow soil microorganisms:

1)oxygen conc

2)pH

3)Temp

4)Pressure'

5)Osmolarity

6)types of nutrients

Bacterial growth caused by what?

Growth in size, needs expansion of cell wall

Septal zonal growth vrs Polar growth in bacteria

• Septal; symmetrical cell growth both sides grow equally

• Polar: Asymetrical growth

Binary fission is what?

• also known as vegetative growth

• Growth in numbers during exponential growth 2^n 1 becomes 2 becomes 4 becomes 8 ect

• replication of chromosome and seperation of cells cells start splitting b4 chromosome duplicates

• DNA replication continues bidirectionally

• 1) Dna origin replicates and migrates

• 2) Dna replication continues bidirectionally

• 3)DNA starts next round

• 4)Septum forms in middle

• 5) division of two cells

Bacterial growth curve?

• log means its doubling : log makes it straight

• stationary phase it is not increasing or decreasing bc not enough nutrients

• steeper the curve faster the growth

What is wrong w this curve?

• not doubling

• growing slow

Interpret this?

the light gray is actually steeper and therefor faster

What is a chemostat and what does it do?

• Chemostat tech ensures logarithmic growth by constantly adding and removing equal amounts of culture media by feeding food and excreting the waste

• make the culture continuous keeps the bacteria at log

• the log OD vs time for a chemostat will have no death phase

Why would bacteria form spores?

• spores can survive non favorable enviorments its a way to keep living

What phase of growth does spore formation occur at?

-stationary

What are the three Types of spores?

1) Endospores: can withstand harsh conditions, produced by bacillius, 1 bacterium produces 1 endospore that gernminates to form 1 bacterium( not reproductive)

2)Asexual reproductive spores (arthrospores): Acintomycetes family less tolerant then endospores but can still survive harsh conditions, 1 bacterium produce many spores (reproductive) function is reproduce and disperse

3)Myxospores: produced by myxobacteria during nutrient limitation

Endospore cycle?

1) septum forms near one pole (asymetrical) Dna replicates and extends into axial filament

2) septum seperates forespore from mother cell, DNA is pumped through septum until each compartment gets a chromosome

3) Mother cell engulfs forespore surrounding it w second membrane

4)Chromosomes of mother cell disintegrate

5)Forespore develops a cortex layer of peptidoglycan between og forespore membrane and the membrane from the mother cell coat proteins are diposited on outer membrane

6)Dipicolinic acid is synthesised and calcium is incorportaed into the spore coat

7)mother cell releases spore

in sporulation inhibitors are inhibited

What is Clostridium botulinum?

• gram positive anarobic bacteria that forms endospores

• vegitative cells produce botulinum toxin that makes botox

• can contaminate food and survive boiling conditions

How to prevent botulism from home canned foods:

• Acidifying low acid foods .acid less than 4.6 prevents endospore germination

• Pressure canner

Asexual reprosuctive spores (arthospores) vrs Endospores

• Asexual spores mother cell produces many babies light and good for spreading

• Endo mother produces one baby and then dies

64% of natural antibiotic classes produced by?

Actinomycetes

How do arthospores produce antibiotics?

• during stationary phase of growth

• What is the basis of Streptomyces Antibiotic Activity Medium (SAM) and Bennet Agar?

• Arthrospores

• moderate amount of carbon and nitrogen induce nutrient limiting conditions driving the culture ton a stationary phase and therefore inducing antibiotic production.

• 
  

• limiting nutrients lead to what?

• lead to spore formation

Do spores produce antibiotics?

no its the vegatative cells that produce antibiotics during the stationary phase

When are Myxospores produced?

When nutreints are limited

How do myxospores function?

• Travel in packs and spirolate when out of food meaning that they put the spores on the inside and let bacteria be on the outside to sacrifice it as food in a spiral

• one cell makes one spore not reproductive

Who is the predator of the microbial world?

Myxobacteria

also produce antibiotics to kill prey

What is the mordant in gram staining?

Iodine

Which bacteria does reproductive sporulation?

What are the two types of microbial nutritional requirements?

1) Macronutrients (g/L, mg/L)

2) Micronutrients (ug/L)

What are examples of Macronutrients?

SCHNOP

Sulfer-in some AA

carbon, hydrogen, oxygen: major biological molecules, carbohydrates,proteins,lipids,nucleic acids

nitrogen: proteins and nucleic acids

phosphorus: nucleic acids and phospholipids

Sources of carbon?

1) Organic carbon sources (heterotrophs)

2) Carbon dioxide CO2(autotrophs): include photosynthetic bacteria eg cyanobacteria chemolithotrophic bacteria

In a lab what can be done to CO2?

• can be bubbled into media

• provided in form of carbonate or bicarbonate salts: CaCO3, NaHCO3 which are converted to CO2 by the enzyme carbonic anhydrase produced by autotrophs

Sources of nitrogen?

1) Ammonium (NH4) or organic nitrogen compounds eg proteins

2) Diazotrophs: few types of bacteria that produce an enzyme called nitrogenase that converts nitrogen gas to ammonium

N2 + 3H2→ 2NH3

Do you need a nitrogen source in media to grow diazotrophs?

no

Examples of Macronutrients?

• Inorganic

• Metal ions: Potassium, Calcium, Magnesium,Sodium,Iron

• CPISM

What are macronutrients used for?

• Required in some enzymes (cofactors)

• Electron carriers

• membrane stability

Examples of Micronutrients?

• in ug/L

• Metal ions: copper, Maganese,Molybdenum,Zinc,Copper,Nickel,Chromium,Selenium,Tungsten

Do all bacteria need micronutrients?

no-only some enzymes need them to function

Examples of supplementary nutrients?

• Vitamins, AA, nucleotides

• may be needed for some microrganisms

What is Bioremediation?

• using living organisms usually microorganisms to clean up waste

• ex Exxon valdez spill 11 million gallons of oil in Alaska bacteria can digest some oil

• crude oil mix of hydrocarbons water insoluble(hydrophobic) lack other macronutrients

• microorganisms can use hydrocarbons from crude oil as a carbon source for growth

What is Bioaugmentation?

• use of fertillizers containing Nitrogen and phosphorous nutrients to ENHANCE growth of microorganisms by providing nutrients that are limited in their enviroment

• can be used to enchance bioremediation of enviormental pollutants by bacteria

Why do different bacteria have different nutrient requirements?

Because of differences in:

(1) metabolic capabilities — the enzymes and pathways they have

(2) their ability to transport a given nutrient into the cell.

What allows cyanobacteria (photosynthetic bacteria) to be autotrophs?

They possess the necessary proteins/enzymes to assimilate carbon in the form of carbon dioxide gas (CO₂).

How do heterotrophic bacteria acquire carbon, and why?

They acquire carbon from organic sources, such as sugars, because they lack the proteins/enzymes needed to fix CO₂ the way autotrophs do.

Why can some bacteria not use lactose as a carbon source?

Because they lack the enzymes needed to transform lactose into central metabolites (usable metabolic intermediates), even if lactose is present.

Besides having the right enzymes, what else does a bacterium need in order to use a nutrient?

The ability to transport that nutrient across the cell membrane and into the cell

What are the three ways culture media can be classified?

(1) physical state: liquid vrs semi-solid

(2) chemical/nutritional content: defined vrs complex, enriched vrs oligotrophic

(3) function (what they're used for) general vrs selective vs differntial vrs enrichment media

What are the two main categories of liquid culture?

(1) Batch culture (tube, flask, etc. with a finite volume of media)

(2) continuous culture.

What is semi-solid media typically composed of, and how is it usually presented?

Media containing agar or another solidifying agent, usually poured into petri plates.

streak plate, pour plate, spread plate

most bacteria cant use agar (from seaweed) as carbon source

What kind of bacterias can grow in solid vrs liquid?

• liquid will have arrobic on top and ararobic on bottom has layers

• solid will only be arobic

What is agar?

• A polysaccharide (made of galactose analogs) derived from the cell wall of red algae.

Why is agar useful as a solidifying agent for bacterial culture media?

• Most bacteria cannot break down this polysaccharide, so they can't use it as a carbon source — it stays structurally stable while bacteria grow on/in it.

• Has thermo-reversble properties It can switch between liquid >85C (random coils) and solid <45C states( helices bundle and bunches form a mesh w water to make gel) depending on temperature, and this process can be repeated (melted and re-solidified).

Increased conc amounts of Agar make the media what?

• more solid

• decreased amounts make it more liquid

What is Gellan gum? (Gelrite)

• another gelling agent

• polysaccaride produced by bacterium Sphingomonas elodea

• can be used to culture thermophiles

Agar and Gellan gum are used in what industry?

• food as thickening agents

• vegan

To obtain high densities of bacterial cells what media would u use?

• liquid bc all parts can support growth anerobic and arobic

To seperate diff species of bacteria in a mixed culture what media would u use?

• solid dont want them to move freely

What is chemically defined media?

• precise composition is known

• you can calculate conc of each element ex C,N.P

What is complex media?

• has macro and micronutrients but exact composition of each element is variable

• batch to batch variation is possible

• ex nutrient broth: beef extract, peptone

Common ingredients of complex media?

Ingredients commonly found in complex media:
Peptone – digests of animal tissues.
Tryptone – enzymatic digest of casein (milk protein).
Yeast extract – cell contents of yeasts.
Beef extract – dried extract of beef (bovine skeletal muscle).
Soytone –enzymatic digest of soy.

Is complex or defined media more time consuming and has more components?

defined

Which medium will allow you to modify eg change the carbon or nitrogen source
without changing the other components ?

defined

In complex media can you swap the individual chemical components (C, N
etc) found in beef extract, peptone, soytone etc?

no

What is enriched media?

• type of complex medium for cultivating fastidious (extra complex) heterotrophs

• - Usually contains blood or serum

What are fastidious heterotrops?

• Fastidious heterotrophs are prokaryotes with complex growth factor requirements
  require additional nutrients not found in normal complex media.

Examples of enriched media agar?

• blood agar:(sheep blood) grows Streptococcus Pyogenes

• Chocolate agar (boiled blood): Neisseria gonorrhea

What kind of agar is blood agar?

complex

fastidious

Heat with agitation to dissolve agar. Autoclave 15 min at 121°C. Cool to 50°C.
Add 5 ml defibrinated sheep red blood cells to 100 ml melted agar.

Does presence of tryptone and phytone/soytone provide enough nutrients for fastidious bacteria?

no

How do you make chocolate agar?

1.Sterillize by autoclaving
2. Add 5-7% v/v of defibrinated blood (horse or
sheep blood). Then place the media in a water
bath of 75 -80°C. Keep swirling gently until the
color changes to dark brown.

hemoglobin solution will be added

Is blood agar only suitable for growth of S. pyogenes ?
For example, do you think Escherichia coli can grow on blood agar ?

yes extra nutrients

Examples of enriched media?

brain heart infusion media

What are oligotrophs?

Bacteria that prefer to grow in media containing lower levels of nutrients, and that normally live in nutrient-poor environments (e.g., sea water).

What growth characteristic is typical of oligotrophic bacteria?

• They are very slow growing.

What example illustrates oligotrophic bacterial growth preferences?

• Some bacteria grow on 1/10 diluted tryptic soy broth (0.1 TSA) but not on full-strength (normal) tryptic soy broth.

• Because oligotrophic bacteria are adapted to low-nutrient environments — high nutrient concentrations (like full-strength media) can actually be inhibitory or unsuitable for their growth, rather than beneficial.

What is general purpose media?

useful for different heterotrophs

may contain extracts and digests of animals, plant or yeast organic matter

What is selective media?

• Selects for growth of organisms with a particular trait

• normally contains compunds that inhibit growth of certain organisms therefore selecting for bacteria that can tolerate the inhibitory compound or condition

What is differential media?

• Allows for visualization of physiological traits usually contains some kind of dye or pH indicator

• due to visible differences in color

What kind of media is macconkey agar?

• Selective and differential

• Complex

• selectes for enteric bacteria

• Crystal violet - kills gram positive bacteria

• Bile salts - allow only bacteria that grows in the intestine
  to survive

• pH indicator - bacteria that ferments lactose form red colonies

• Lactose – main carbon source

• Peptone – provide other macro and micronutrients

• Agar - to solidify media

Why is MacConkey agar selective?

• MacConkey agar is selective because it contains bile salts and crystal violet, which inhibit the growth of Gram-positive bacteria (like S. aureus) while allowing Gram-negative bacteria (like E. coli) to grow

How is MacConkey Agar diifferential?

• MacConkey agar is differential because it contains lactose and a pH indicator — lactose-fermenting bacteria (like E. coli) produce acid that turns their colonies pink, while non-lactose fermenters (like S. typhi) can't ferment it and stay white, letting you visually distinguish between the two even though both can grow.

What is enrichment media?

• Use to increase the concentration of one microbe over others, therefore
  enriching a sample for the microorganism of interest.

• Generally a liquid media.
  Include specific nutrients/compounds to promote growth of particular bacteria.

• Unlike selective media, it rarely contains inhibitory compounds to prevent
  growth of certain organisms.

What is the key difference between enrichment media and enriched media?

• Enrichment media increases the relative abundance of a target organism from a mixed sample (usually liquid, no inhibitors)Encourage growth of some microorganisms SUPPRESS growth of others. We are increasing the concentration of particular microbes in this medium – hence enrichment
  of particular microbes growth of one kills other bc too good

• enriched media supports growth of fastidious organisms by adding extra nutrients (like blood or serum) to complex media. make richer leave other alone

What is the purpose of using M9 salts + crude oil as a medium?

To enrich for bacteria from soil that can use crude oil as a carbon source — since M9 salts alone provide no organic carbon, only bacteria capable of metabolizing hydrocarbons in crude oil can grow, allowing them to be selectively enriched from a mixed soil sample.

Examples of enrichment media

• Alkaline-Peptone water

• Enrichment media for Vibrio spp. Vibrio can tolerate high
  salt concentration and alkaline pH.
  Can be used to promote growth of Vibrio in a mix culture sample e.g.
  wastewater.

What is an unculturable microorganisms?

• Bacteria that can only be cultured in living hosts ex: Mycobacterium leprae causes leprosy

• more than 99% of bacteria present in environment cant be cultured