Section 2 final draft microbio lab midterm

list the nutritional needs of microbial cells

1. carbon

2. nitrogen

3. nonmetallic elements

4. metallic elements

5. vitamins

6. water

7. energy

the most essential and central atom common to all cellular structures and functions

carbon

carbon

the most essential and central atom common to all cellular structures and functions (carbohydrates- glucose or lactose)

what are the two carbon-dependent types of microbial cells

autotrophs and heterotrophs

organisms that can be cultivated in medium consisting of solely inorganic compounds; they use inorganic carbon in the form of CO2

autotrophs

autotrophs

1. category

2. define

1. one of the two types of carbon dependent types of microbial organisms

2. organisms that can be cultivated in medium consisting of solely inorganic compounds; they use inorganic carbon in the form of CO2

organisms that cannot be cultivated in a medium consisting solely of inorganic compounds; they must be supplied with organic nutrients, primarily glucose

heterotrophs

heterotrophs

1. category

2. define

1. one of the two types of carbon dependent types of microbial organisms

2. organisms that cannot be cultivated in a medium consisting solely of inorganic compounds; they must be supplied with organic nutrients, primarily glucose

an essential atom in many cellular macromolecules, particularly proteins and nucleic acids

nitrogen

what are the major nonmetallic ions used for cellular nutrition

sulfur, phosphorous

sulfur and phosphorus

the major nonmetallic ions used for cellular nutrition

integral to some amino acids, and a component of some proteins; sources include organic compounds such as —- containing amino acids, inorganic compounds, such as sulfates, and elementary—-

sulfur

necessary for the formation of the nucleic acids and for synthesis of high-energy organic compound adenosine triphosphate (ATP) also supplied in salt form

phosphorous

give some metallic elements

CA2+, Zn2+, Na+, K+, CU 2+, Mn 2+, Mg 2+, Fe 2+, Fe 3+

necessary for efficient performance of varied cellular activities such as osmoregulation, regulation of enzyme activity, and electron transport during bio-oxidation. Micronutrients and required in trace concentrations only; inorganic salts d

metallic elements

organic substances contribute to cellular growth and are essential in minute concentration for cell activities. Also sources of co-enzymes, which are required for the formation of active enzyme systems. Some microbes require these to be supplied in a performative state for normal metabolic activities. Some possess extensive synthesizing pathways, whereas others can synthesize only a limited number from the other compounds present in the medium.

vitamins

all cells require this in the medium so that lower molecular weight nutrients can cross the membrane

water

why is water a nutritional need

all cells require water in the medium so that lower molecular weight nutrients can cross the membrane

active transport, biosynthesis, and biodegredation of macromolecules are the metabolic actives of cellular life. These activities can be sustained only if there is a constant availability of within the cell

energy

use radiant energy as their sole energy source

phototrophs

chemotrophs

depend on the oxidation of chemical compounds as their energy source, use organic compounds like glucose, or inorganic compounds like H2S or NaNO2

what are the abiotic/physical factors that influence microbial growth?

1. temperature

2. pH

3. gaseous requirement / atmospheric oxygen

explain how temperature affects the growth and survival of cells

influences the rate of chemical reactions

low temp= slow/inhibits enzyme activity, inhibiting metabolism and cell growth

high temp= coagulation and can irreversibly denature thermolabile enzymes

enzymes differ in terms of heat sensitivity, but generally anything in the range of 70C will destroy bacterial enzymes—> cell death

explain how pH affects the growth and survival of cells

effects cells enzymatic activities

optimal pH = 7

increase in H+ ions= lower pH= more acidic pH

decrease in H+ ions = higher pH= more alkaline conditions

an increase, or decrease in pH= detrimental; will slow down rate of chemical reactions due to destruction of cellular enzymes

explain how atmospheric oxygen/gaseous requirement affects the growth and survival of cells

most cells need atmospheric oxygen for respiration. Atmospheric oxygen—> ATP formation and the availability of energy in a utilizable form form for cell activities; some cell types lack the enzyme systems for respiration in the presence of oxygen, so they have to use an anaerobic form of respiration or fermentation

How do you name a bacteria/ microorganism?

First letter of genus= capitalized, entire name of species (lowercase) follows

Composition

The composition of a growth medium can vary greatly depending on the organisms that will be grown on it. Medium is generally categorized as either defined or complex

Gram positive microorganisms (6)

1. S. aureus

2. S. epidermidis

3. E. faecalis

4. B. stearothermophilus

5. C. sporogenes

6. M. Luteus

Gram negative bacteria (5)

1. E. coli

2. K. aerogenes

3. P. aeruginosa

4. S. marcescens

5. P. fluroescenes

Fungi (2)

1. Saccharomyces cerevisiae

2. Aspergillus niger

Bacterial growth rate phases (4)

lag phase, log phase, stationary phase, death phase

lag phase of bacterial growth

bacteria adapt themselves to new growth conditions; where the synthesis of RNA, enzymes, and other molecules begin

log phase of bacterial growth rate

characterized by cell doubling at a constant rate (exponential phase)

stationary phase of bacterial growth rate

bacteria stop growing, often due to nutrient limitation; growth and death rate are roughly equivalent

death phase of bacterial growth rate

bacteria die, usually due to adverse conditions; aka decline phase

metabolism

the sum of biochemical reactions required for the generation of energy and the utilization of energy and the utilization of energy to produce cellular components

metabolism

during active growth, bacteria must be undergoing constant —-

Catabolism

energy generation

anabolism

biosynthesis using energy

chemotrophs

organisms that get their energy from something other than sunlight like chemical reactions; gets carbon source from inorganic molecules or from the breakdown of food

autotrophs

a type of chemotroph that gets its carbon source from inorganic molecules like CO2 ,H2O; do not depend on another organism or chemical for their food, produce their own food

heterotroph

a type of chemotroph that gets its carbon source from the breakdown of food; requires organic material; can only break down food

chemoheterotrophs

MOST BACTERIA, especially most pathogens are classified as this (based on where their energy comes from and how they use it

Microbial requirements *= culture media

carbon, nitrogen, nonmetallic elements (S and P), metallic elements, vitamins, water, temperature*, pH of environment, atmospheric oxygen**

two ways the composition of a growth medium can be referred to as

Define and complex

Chemically defined media

These are composed of known quantities of chemically pure, specific organic and/or inorganic compounds. Their use requires knowledge of the organism’s specific nutritional needs

defined media aka

synthetic

what things can you find in synthetic/ defined media

carbon/ energy source

inorganic nitrogen (ammonium phosphate)

salta (NaCl, CaCl2, MgSO4)

growth factors specific to organism- amino acids, vitamins, etc.

complex media aka

undefined media

Complex Media

These are composed of a number of complex substances, plant and animal extracts, whose exact chemical compositions are not known. They can support the growth of most heterotrophs

a type of complex media

enriched media

Enriched media

a subset of complex media that allows for the cultivation of fastidious microorganisms, organisms that have highly elaborate and specific nutritional needs. These bacteria may not grow or grow poorly on basic artificial medium and require the addition of one or more growth-supporting substances such as additional plant or animal extracts, vitamins, or blood.

what can you find in complex (undefined media)

trypsin breakdown of proteins (peptone)

vitamins

carbohydrates (glucose)

salts/buffers

additional supplements (blood, milk, etc)

what are the types of complex media we use

nutrient broth/agar

tryptic soy broth/agar

lysogeny broth (LB)/ agar

components of media

Types of media

general growth media, selective (enrichment) media, differential media

general growth media, selective (enrichment) media, differential media

all of these can be either complex or defined

General growth media

non-specific media configured to culture a wide range of microorganisms without many restrictions

what are examples of general growth media

nutrient agar and luria- bertani (LB) broth

selective (enrichment) media

1. define

2. usage/purpose

3. example

1. one of the types of media

2. used to isolate microorganisms in a mixed culture, allows for the growth of particular microorganisms and restricts others using inhibitor components

3. Pseudomonas Isolation Agar (PIA)- isolate and cultivates Pseudomonas while restricting growth of most contaminating organisms

what role do bile salts, crystal violet and an antibiotic play in selective media?

allows it to inhibit growth of certain microorganisms

what are some growth inhibiting microorganisms found in selective media?

bile salts, crystal violet, and antibiotics

differential media

1. define

2. usage/purpose

3. example

1. a type of media

2. contains chemical compounds that cause observable change in the medium or the medium surrounding the bacteria when a specific reaction occurs, allowing you to decipher between cells; distinguishes microorganisms based on their morphology and biochemical processes

3. fermentation of sugars, pH indicators, blood agar

blood agar

a type of differential media that will

selective agents

inhibit/ prevent growth of undesired organisms; bile salts, high salt concentration, dyes, antibiotics

differential reagents

•Often pH indicator dyes

•The allows us to detect acidic/basic byproducts of metabolism when bacteria grow using specific substrates.

•These differences in metabolism are often unique to certain species of bacteria, and well-conserved among all isolates of a species.

•Different species of bacteria have adapted to prefer different food sources, and produce different byproducts. We can exploit these preferences!

why do media change color?

color change occurs when microorganisms metabolize a substrate (often a sugar), producing metabolic byproducts such as acids (ex: ferments lactose, produces lactic acid, acid lower pH, pH indicator dye changes color

Mannitol Salt Agar

1. Purpose

2. selective agent

3. differential agent

4. explain interpretations of it

1. used to test for the presence of Staphylococci

2. selective for Staphylococcus species; selective agent= High salt concentration (7.5%) NaCl; selective against non-staphylococci species like E.coli and other Gram negative bacteria (won’t grow because of the salt)

3. differential for Staph. aureus; mannitol is fermented by S. aureus and produces acid; phenol red is the pH indicator, which turns yellow, indicating mannitol fermentation (aka presence of S. aureus. other staph species like S. epidermidis can grow on mannitol salt agar, but will not show any yellow around it (doesn’t ferment the mannitol)

explain how mannitol fermentation relates to pH

when the mannitol is fermented (which it will be by S. aureus), it has a byproduct of acid, this acid causes the pH to lower, causing a change in the phenol red to change to yellow, once the pH is below 6.8

phenol red

1. what value is it red at? & what does it mean

2. what value is it yellow at? & what does it mean?

1. above 7.4 (meaning that there’s no mannitol fermentation, and therefore, no presence of staph. aureus

2. below 6.8 = positive for S. aureus

given the picture, give me the interpretations

top: S. aureus present (indicated by bacterial growth and yellow change in phenol red pH indicator)- mannitol fermentation took place, pH must be below 6.8- positive result

left: staph species present - growth, but phenol red= red; pH must be above 7.4

right: species other than staph (gram - specifically) here - no growth and phenol red= red because no acid production (aka no mannitol fermentation)- no result

Blood Agar

1. Purpose

2. differential agent

3. explain interpretations of it

1. used to differentiate bacteria based on hemolysis of red blood cells; tends to recover fastidious bacteria, often Streptococcus species NOT SELECTIVE

2. differential based on hemolysis of RBCs- is it sheep’s blood? and enriched (RBCs)- iron in Hb

3. green halo around bacterial species= alpha hemolysis (strep. pneumonaie); complete zone clearing(looks like its glowing) (strep. pyogenes); no clearing (looks very faint)= no color change= non-pathogenic bacteria

4. hemolytic pathogens (strep and staph)

what are fastidious bacteria

bacteria that require special nutrients or enriched media to grow

what is blood agar made of?/ what purpose does it serve?

blood agar is a trypic soy agar plate which is more general growth medium, but it contains whole sheep’s blood; this allows it to be differential for the ability of bacteria to lyse RBCs

what are the types of hemolysis

alpha hemolysis, beta hemolysis, and gamma hemolysis

partial/incomplete hemolysis, zone of partial clearing= green halo around bacterial colonies

alpha hemolysis (ex: Strep. pneumoniae)

green halo around bacterial colonies indicates what?

alpha hemolysis, can be strep. pneumoniae

complete hemolysis, complete zone of clearing around colonies

beta hemolysis; strep. pyogenes

what does beta hemolysis look like?

looks like a halo, huge zone of clearing around and below the colony

no lysis of RBC, no clearing of medium surrounding colonies, no color change

gamma hemolysis (non-pathogenic bacteria)

no hemolysis;

gamma hemolysis

what does gamma hemolysis look like?

no clearing of the medium surrounding the RBC, looks like normal bacterial growth, no crazy change

label them

explain what the following descriptions mean and what species could be residing there:

1. complete clearing zone around colony

2. partial clearing zone around colony- slightly green

3. no color change in media, surrounding media remains red

1. beta hemolysis (complete lysis of RBC) strep pyogenes

2. alpha hemolysis strep pneumoniae

3. gamma hemolysis (non-pathogenic bacteria

positive blood agar results

negative blood agar results

1. beta hemolysis (complete clearing)

2. gamma hemolysis (no clearing - little to no change)

label them

beta, alpha, and gamma hemolysis, respectively

MacConkey Agar

1. Purpose

2. selective agent

3. differential agent

4. explain interpretations of it

1. selective for gram - bacteria (enterobacteriaecae) and differentiates lactose fermenters vs. non-lactose fermenters***

2. selective for gram negative enteric bacteria (enterobacteriaceae); selective agents; selective agent= crystal violet and bile salts, inhibits G+

3. differential for coliforms (lactose fermenters) vs. non-coliforms (non-lactose fermenters); differential agent= neutral red indicator and lactose

neutral red indicator and lactose

1. what are they

2. explain what they look like? what does this indicate?

1. they’re the differential agents for MacConkey Agar

2. neutral red indicator: if pH is above 6.8= colorless (non-coliforms ; if above 6.8= RED/Pink (coliforms)

what is a coliform?

a coliform is a rod-shaped gram -, non-spore forming bacteria which can ferment lactose with the production of acid and gas when incubated at 35-37C; typically colon bacteria

a rod-shaped gram -, non-spore forming bacteria which can ferment lactose with the production of acid and gas when incubated at 35-37C; typically colon bacteria

coliform

bacteria that do not ferment lactose

non-coliform bacteria

non-coliform bacteria

bacteria that do not ferment lactose

how are bile salts and crystal violet selective against G+ bacteria

they don’t have the outer membrane to protect the cell wall (like gram -) bacteria

examples of coliform bacteria

Escherichia, Klebsiella, Serratia

Escherichia, Klebsiella, Serratia are examples of what

coliform bacteria

Salmonella, Shigella, and Yersinia

examples of non-coliform bacteria

Salmonella, Shigella, and Yersinia are all examples of what?

non-coliform bacteria; note that these are still gram - bacteria, so they do still grow on MacConkey Agar

what is the original color of MacConkey Agar

very light pink/ almost light orange looking

explain the picture using technical terms

1. what this showing (what type of agar)

2. explain whats happening on the right and what it can be

3. explain what’s happening on the left and what it can be

1. MacConkey Agar

2. non-lactose fermenting colonies, no lactic acid formation, pH is above 6.8, so indicator dye is colorless= light orange-ish color; Salmonella, Shigella, or Yersinia

3. lactose fermenting colonies, lactic acid byproduct—> everything turns light pink; E.coli, Klebsiella, Serratia

describe the following, provide the mechanism behind it, and give an example of what type of organism it can be:

1. Positive result for MacConkey agar

2. Negative Result for MacConkey agar

red/pink color, coliform aka lactose fermenters are present,

light orange/ colorless, non-coliform, non-lactose fermenters present