MCB 2610 Exam 2

Alcohol

(ethanol) - used in lab settings and present in most hand sanitizers · among the most widely used disinfectants and antiseptics · two most common are ethanol and isopropanol (60-80%) · bactericidal, fungicidal, but not sporicidal (cannot use alcohol-based hand sanitizer if you are concerned about spreading things like C diff.) · inactivate some viruses · denature proteins and possibly dissolve membrane lipids

oxidizing agents

(sodium hypochlorite) commonly added to swimming pools and hot tubs to inhibit microbial growth

Micronutrients

(trace elements) o Mn, Zn, Co, Mo, Ni, and Cu o Required in trace amounts o Often supplied in water or in media components o Ubiquitous in nature o Serve as part of enzymes and cofactors o Aid in catalysis of reactions and maintenance of protein structure

phenolic compounds

- (triclosan) - added to numerous products, including some soaps, deodorants, and cosmetics · Phenol was 1stwidely used antiseptic and disinfectant. Used in 1867 by Joseph Lister · commonly used as laboratory and hospital disinfectants (e.g., Lysol) · act by denaturing proteins and disrupting cell membranes · tuberculocidal, effective in presence of organic material, and long lasting (can kill Myobacterium tuberculosis, which is very difficult to kill) · disagreeable odor and can cause skin irritation · E.g., triclosan is used in hand sanitizers (not allowed in products right now because used so much that bacterial resistance is a fear) (in Colgate toothpaste because it prevents cavities)

Where did they source their leech isolates

- FDA supplier of medicinal leeches in CT - Clinical Isolates from Patients in France, CA, IA, Idaho - Farms in France (and Germany)

small subunit rRNA

- Most widely used gene in constructing phylogenetic trees. - sequence variable regions and amplify to learn about members of microbial community

Can microorganisms change nutritional type?

- Some organisms have great metabolic flexibility and alter their metabolism in response to environmental changes (ex. When oxygen is limiting they can switch their metabolism to use light as source of energy -> facultative anaerobe (E. coli) - This provides distinct advantages to organisms that experience frequent changes to environmental conditions

The Turbidostat

- another type of continuous culture system - Photocell - regulates the flow rate of media through vessel to maintain a predetermined turbidity or cell density - You set the flow rate - dilution rate varies - no limiting nutrient - turbidostat operates best at high dilution rates

Autoclave

- apparatus for sterilizing by steam under pressure - cannot put plastic petri dishes in autoclave unless looking to destroy them

The Chemostat

- most common type of continuous culture device - A chemostat flows in fresh medium and takes out some old medium/microbes to keep the culture in continuous operation. - rate of incoming medium = rate of removal of medium from vessel - an essential nutrient is in limiting quantities

Suppose hospital custodians have been assigned with cleaning all showerheads in patient rooms to prevent disease spread. What factors would have the greatest impact on effectiveness of disinfectant used?

-Need to worry about temperature (shower has hot water)-pH of the water -Think about the source of the water; down stream up stream?-Think about disease you want to kill -Risidual disinfectants (are any cleaners toxic)-Longer contact time (remove shower head and soak it)

psychrophiles

0 C - 20 C

psychrotrophs

0 C - 35 C organisms that cause food spoilage in the refrigerator

3 major classes of growth factors

1. Amino acids: Needed for protein synthesis 2. Purines and pyrimidines: Needed for nucleic acid synthesis 3. Vitamins: Small organic molecules that function as enzyme cofactors, needed in very small amounts

Bacterial Colony Morphology

1. Form 2. Elevation 3. Margin

Factors affecting microbial growth

1. Nutrient Concentration 2. Effects of oxygen on microbial growth

Functional Types of Media

1. Supportive or general-purpose media 2. Enriched media 3. Selective media 4. Differential

Conditions Influencing the Effectiveness of Antimicrobial Agent Activity

1. population size o larger populations take longer to kill than smaller populations 2. population composition o microorganisms differ markedly in their sensitivity to antimicrobial agents o ex. Spores are very resistant (c diff harder than e coli) 3. concentration or intensity of an antimicrobial agent o usually higher concentrations or intensities kill more rapidly (not always, e.g., EtOH) àethanol needs to be greater than 60% (very volatile so % decreases over time); higher concentrations like 100% is also ineffective o relationship is not linear 4. Contact time - duration of exposure o longer exposure = more organisms killed 5. temperature o higher temperatures usually increase amount of killing 6. local environment o many factors (e.g., pH, viscosity, and concentration of organic matter) can profoundly impact effectiveness o organisms in biofilms (layers of microbes) are physiologically altered and less susceptible to many antimicrobial agents = may kill top layer but not penetrate all the way through

If a single bacterium reproduced every 20 minutes, how many would there be after 2 hours? How many generations is that?

120/20 = 6 1 - 2 - 4 - 8 - 16 - 32 - 64 6 generations

mesophiles

20 C - 45 C

what is the acceptable colony range?

30-300

thermophiles

55 C - 85 C

Hyperthermophiles

85 C - 113 C

How does a continuous culture system differ from a batch culture system?

A Batch culture is a closed laboratory system that has a characteristic pattern of growth with distinct phasesThere are no nutrients, cells, or wastes coming in or out: nutrients are only added in the beginningA continuous culture has the same amount coming in and out(turbitostat and chemostat) Nutrients can be added. Growth can be controlled more. Environment is constant. High turnover rate. Closer to natural environment not industrial production. Not as easy to set up. More chance of contamination.

GasPak jar

A container used to incubate obligate anaerobic organisms

How do you measure microbial population growth if cells are already very diluted?

A filter apparatus can concentrate the cells - Membrane Filtration Method: MUST BE LIQUID

Anaerobic Chamber

An enclosed compartment maintained in an anaerobic environment; used to grow strict anaerobes in lab

Extracts

Aqueous extracts, usually beef or yeast

What are the CFU/mL values at 1, 2, and 3 hours, calculated using the numbers listed here?

Average the two samples together, add a degree because it has 0.1 mL so extra dilution (won't be like this on the exam) 1 hr: 8.5 x 104CFU/mL 2 hr: 1.845 x 105CFU/mL 3 hr: 6.3 x 105 CFU/mL

When using the pour plate or spread plate method of determining bacterial counts, why are the results expressed as CFU's and not as number of cells?

Because a colony can have a number of cells even though it looks like one

Why is it difficult for organisms to grow at low aw?

Because they would have to pull the water out away from the solutes and that takes energy (too energy intensive)

What elements make up carbohydrates?

C, H, O

What elements make up nucleic acids?

C, H, O, N, P

What elements make up proteins?

C, H, O, N, S

What elements make up lipids?

C, H, O, P

What is the backbone of all organic components present in a cell?

Carbon

What happened to rate behavior when their microbiomes were removed?

Caused rats to become more adventurous

Why do shift-up and shift-down experiments cause cells to go into unbalanced growth?

Cells need to adjust to new conditions before reentering balanced growth

To what major nutritional group do you think human microbial pathogens belong?

Chemoorganoheterotroph

Which method do you thin would give the most accurate measurement of cell number?

Depends on what you want to know, dead or alive, outcome, starting specimen, sample size

Which method would you use to count the number of bacteria in a sample of milk that needs to go out the door ASAP?

Direct Microscopic Count on the counting chamber

In which category do you think most household cleaners would belong to?

Disinfectant

Chemical Control Agents

Disinfection Antisepsis Sterilization

Why do organisms need electrons?

Electrons play a role in energy production used for cellular work and reduction of Co2 to form organic molecules

What would be the best method to sterilize plastic Petri dishes?

Ethylene Oxide Sterilizer

What are the advantages and disadvantages of using gamma radiation to sterilize materials?

Expensive, need certifications, effective, penetrates deep within so works great with all kinds of food products

What bacteria was more abundant on the hands of women than men?

Lactobacilaece

Practical issues for destroying microbes or preventing their growth

Measuring effectiveness

Why is it so difficult to grow some organisms in a laboratory culture?

Must meet nutritional requirements to grow, but you might not know what the necessary growth factors are

Is a colony formed as a result of streaking a plate always derived from a single bacterium? (the great plate count anomaly)

No, some organisms grow in clumps or long chains, so those would be very difficult to isolate a single cell.

Eosin Methylene Blue Agar (EMB)

Organism identified: Gram-negative enterics (Enterobacteriaceae) Selective: medium partially inhibits Gram-positive bacteria Differential: Eosin and methylene blue differentiate among organisms: E. coli colonies are purple and typically have metallic green sheen; Enterobacter aerogenes colonies are pink, indicating that they ferment lactose; and colonies of other organisms are colorless, indicating that they don't ferment lactose

Brilliant Green Agar

Organism identified: Salmonella Selective: brilliant green dye inhibits Gram-postive bacteria and thus selects for Gram-negative ones Differential: Differentiates Shigella colonies ( which do not ferment lactose or sucrose and are red to white) from other organisms that do ferment one of those sugars and are yellow to green

What would happen to the dynamics of a chemostat if the sterile medium being added gets contaminated?

Organisms will be competing with each other for the nutrients.

What are some less common growth factors?

Other less common growth factors include heme (cytochromes, Haemophilus influenze) and cholesterol (required for some mycoplasmas)

Peptones

Protein hydrolysates prepared by partial digestion of various of various protein sources

A large hospital washes burn patients in a stainless-steel tub. After each patient, the tub is cleaned with a Quat. It was noticed that 14 of 20 burn patients acquired pseudomonas infection after being bathed. How would you explain this high rate of infection?

Quaternary ammonium compounds are most effective against gram-positive bacteria. Gram-negative bacteria that were stuck in cracks or around the drain of the tub would not have been washed away when the tub was cleaned. These gram-negative bacteria could survive the washing procedure. Some pseudomonads can grow on quats that have been accumulated.

What bacteria was more abundant on the hands of men than women?

Staphylococci

Agar

Sulfated polysaccharide used to solidify liquid media; most microorganisms cannot degrade it - usually extracted from red algae

Why are peptones, yeast extract and beef extract added to growth media?

This makes it complex medium. We do not know the growth requirements, so these are added in as all purpose components

Superoxide anion (O2-)

Toxic oxygen species cellular defense: superoxide dismutase, superoxide reductase enzymes

Hydroxyl radical (OH)

Toxic oxygen species sources: by-products of reduction of O2 during respiration and other biochemical redox reactions cellular defense: antioxidants such as glutathione

Complex Media

Unknown chemical composition - nutrient broth - Tryptic soy broth - MacConkey Agar

What are the advantages and disadvantages of using UV light to sterilize materials?

Very inexpensive, effective, does take some time, doesn't penetrate

Pure culture

a population descended from a single cell

Which physical or chemical reagent would be the best choice for sterilizing: a) glass pipettes b) tryptic soy broth tubes c) nutrient agar d) antibiotic solutions e) interior of biological safety cabinet f) wrapped package of plastic petri plates explain

a) autoclave - glass does not melt b) autoclave - heats growing media until bacteria, viruses and spores are all killed c) autoclave - heats growing media until bacteria, viruses and spores are all killed d) ionizing radiation - good penetration and kills bacteria and spores; does not inactivate antibiotics or their potency of (autoclave destroys antibiotic) e) UV radiation or sterilizing gas - easily sterilizes air/surfaces w/in the cabinet; does not effect non-living surfaces f) ionizing radiation or ethylene oxide - capable of penetrating plastic & other substances (plastic cannot be autoclaved)

Nutritional Requirements of microorganisms: Macronutrients

all cells need access to large amounts of carbon, nitrogen, phosphorous, sulfur, and oxygen to build macromolecules

3. Autotrophs

assimilate carbon from inorganic sources

3. Heterotrophs

assimilate carbon in preexisting organic form

No matter what an organism's nutrient requirement they require a ____________________

balanced mix

1. Chemotrophs

capture energy from oxidation of reduced organic or inorganic compounds

1. Phototrophs

capture light energy to produce ATP

If a product says it kills 99.99% of germs, is that good?

depends on the starting count and type of microbe if 10^6starting count 99.99% killed = 100 cells infective dose of Listeria monocytogenes= 1000 cells infective dose of Salmonella enterica = 10-100 cells infective does of vibrio cholera= 100 cells

Both spread and pour plate used to

determine the number of viable microbes in an original sample and have to ensure both are given proper growth conditions

What is the best method to measure microbial growth in a urine sample?

dilution-incubation

Generation time

doubling time o time required for the population to double in size o varies depending on species of microorganism and environmental conditions o range is from 10 minutes for some bacteria to several days for some eukaryotic microorganisms

What is the best method to measure microbial growth in a drinking water sample?

filtration concentration? turbidity?

Would UV radiation or gamma radiation be better for sterilizing ground beef?

gamma

Anaerobes

grow in the absence of oxygen - Aerotolerant anaerobes aren't harmed by oxygen but don't use it either - Obligate anaerobes cannot grow when oxygen is present - Facultative anaerobes can use oxygen but can also grow in the absence of oxygen

Aerobes

grow in the presence of oxygen - Obligate aerobes REQUIRE oxygen - Microaerophiles grow best when there is less oxygen than normal - Often determined by what defenses are available against oxygen's negative effects in the cell

Along with carbon, what elements are also found in organic molecules

hydrogen and oxygen

Increase in cellular constituents may result in

increase in cell number increase in cell size

-static agents

inhibit the growth of microorganisms · -static o suffix indicating that agent inhibits growth o include bacteriostatic and fungistatic

-cidal agents

kill microbes o suffix indicating that agent kills o germicide: kills pathogens and many nonpathogens but not necessarily endospores · include bactericides, fungicides, algicides, and viricides

Benzalkonium chloride

major ingredient in Lysol

Direct Counts

o A special slide with an etched grid can be used o A known volume is loaded onto the grid and the cells are counted under a light microscope o Pros: cheap, fast, easy o Cons: you can't differentiate living vs dead o Use this method for milk

Turbidity

o A spectrophotometer sends light through a culture o If tube is cloudy, light won't get through the tube and strike the unit's sensor (high absorbance) o It can give a rough measure of cell density in the tube

Unculturable bacteria: what if it won't grow?

o Cultivation independent methods - DNA from unculturable bacteria can be amplified and sequenced by PCR (polymerase chain reaction) - Sequences can be used to produce fluorescent probes that will bind to complementary DNA (fluorescent in situ hybridization - FISH) o Some microbes may just be too accustomed to growing with their friends and neighbors to be isolated

Antisepsis

o Destruction or inhibition of microorganisms on living tissue o antiseptics: chemical agents that kill or inhibit growth of microorganisms when applied to tissue o cannot damage tissue

How can we measure and count microbes?

o Direct measurements - Microscope counts - Viable (living) cell count on solid medium o Indirect measurements - Dry weight - Turbidity (cloudiness) - Chemical analysis

Differential Media

o Distinguish between different groups of microorganism based on their biological characteristics - Blood agar · Hemolytic vs nonhemolytic bacteria - MacConkey agar · Lactose fermenters vs nonfermenters

Selective media

o Favor the growth of some microorganisms and inhibits growth of other - MacConkey agar - selects from gram-negative bacteria - Useful in things like water treatment and food processing to prevent salmonella or e coli but removal of other organisms is not necessary

Physical removal of microbes by filtration

o Filtration has been a method of purifying liquids for centuries (using sand, charcoal, etc.). o Newer methods use nylon/Teflon filters with a pore size of 0.2 or 0.45 μm (small enough to keep out most eukaryal and bacterial cells). o Viruses can be removed from liquids by ultrafiltration methods (reducing pore size 10 to 100 nm). · Problems can result, though... o Large particles clog filters. o Ultrafiltration requires high pressure. o Viscous liquids don't filter well.

Enriched media

o General purpose media supplemented by blood or other special nutrients o Blood agar: human and animal pathogens grow better in this Streptococcus pyogenes (cause strep throat) -Beta-hemolytic - clearing zone around bacteria because hemolyzed or broken-down sheep's blood o Chocolate agar - Auto plated sheep's blood agar (pressure cooker)

Nutrient concentration and microbial growth

o Growth rate will depend on amounts of nutrients in environment o One key nutrient, available in the lowest amount, will dictate how much growth can occur over time (i.e. it will be a limiting factor) Steeper the slope, the faster the growth rate

Chemical methods of controlling microbes

o Many chemicals can kill microbes, or inhibit their growth. o Disinfectants: Chemicals used on non-living surfaces to kill potentially infectious microbes. o Antiseptics: Chemicals that can be used on living tissue to kill potentially infectious microbes (usually only used topically) -Need to make sure it will not kill living tissue

Why use a continuous culture?

o Microbes in nature don't exist in a closed system. · We may want to keep microbes in exponential growth to harvest one of their products. · We may want to keep microbes in a limited but continuous flow of nutrients to mimic their environmental conditions.

Obtaining a pure culture

o One of the benefits of a solid medium is that cells are held in place on the surface and can be isolated o This can lead to separating a mixture of cells into a pure population o There are three basic methods for separating cells on a plate § Streak plate method - best for isolating pure cultures § Spread plate method - best for quantifying § Pour plate method - best for quantifying

Why is Phosphorus an absolute requirement to microorganisms and how is it supplied?

o P is needed for nucleic acids, phospholipids, nucleotides, some cofactors, some proteins, etc o Usually supplied as inorganic phosphate - Most organisms use inorganic phosphorus which is directly incorporated into their cells

Spread plate bacterial count

o Sample is serially diluted o Small volume of dilute mixture containing approximately 30-300 cells is transferred o Spread evenly over surface with a sterile bent rod

Why is Sulfur an absolute requirement to microorganisms and how is it supplied?

o Sulfur is needed for amino acids cysteine and methionine, some carbohydrates, biotin and thiamine o Usually supplied as sulfate via assimilatory sulfate reduction - Most organisms use sulfate and reduce it by assimilatory sulfate reduction

supportive or general purpose media

o Support the growth of many microorganisms o Tryptic soy agar

Electromagnetic radiation to prevent microbial growth (UV radiation)

o UV radiation of 260 to 280 nm wavelengths can damage DNA, forming thymine dimers. (thymines bind to each other instead of adenine - distorts DNA) o This can be exploited to control microbial growth on non-living surfaces and in water. o UV light is good for surfaces but does not penetrate within

sterilzation

o destruction or removal of all viable organisms

disinfection

o killing, inhibition, or removal of disease causing (pathogenic) organisms o disinfectants - agents, usually chemical, used for disinfection - usually used on inanimate objects o not killing everything o used on fomites (inanimate objects)

Why is Nitrogen an absolute requirement to microorganisms and how is it supplied?

o needed to make Amino acids, purines, pyrimidines, some carbohydrates, lipids, enzyme cofactors, etc. o Supplied in numerous ways such as metabolism of amino acids, nitrates, ammonia, and some fix N2 from atmosphere

Chorine

o oxidizes cell constituents o highly efective o important in disinfection of water supplies and swimming pools, used in dairy and food industries, effective household disinfectant o destroys vegetative bacteria and fungi, o chlorine gas is sporicidal o can react with organic matter to form carcinogenic compounds; a lot of organic matter can tie up the chlorine (ineffective) o E.g., Bleach is highly effective (10% with a little vinegar) (works best at a slightly lower pH)

Membrane Filters

o porous membranes with defined pore sizes that remove microorganisms primarily by physical screening o have replaced depth filters for the most part o Removes microorganisms from heat sensitive liquids (e.g., pharmaceuticals, antibiotics, vitamin solutions, culture media, etc)

Sanitization

o reduction of microbial population to levels deemed safe (based on public health standards) o silverware at a restaurant

Iodine

o skin antiseptic o oxidizes cell constituents and iodinates proteins o at high concentrations may kill spores o skin damage, staining, and allergies can be a problem o E.g., Wescodyne o iodophore § iodine complexed with organic carrier § released slowly to minimize skin burns

chemotherapy

o use of chemicals to kill or inhibit growth of microorganisms within host tissue o internally o i.e. antibiotics

2. Organotrophs

obtain electrons from organic compounds

Glutaraldehyde

often used to prepare biological specimens

Neutrophhiles

pH 5.5-8.5

Acidophiles

pH <5.5

Alkophiles

pH >8.5

growth refers to

population growth rather than individual cell growth

Advantages and Disadvantages of Turbidity

pro: can be used for microbes that cannot be cultured on plate media cons: uses a lot of test tubes; yields the most probable number (possibly less accurate)

Advantages and Disadvantages of direct counts

pros: cheap, fast, easy cons: you cannot differentiate between living and dead

How would you determine if filtering had removed all microorganisms from the air or a solution?

put a plate in the hood and see if something grew there

Membrane Filter Sterilization

removes microorganisms from heat sensitive liquids

Some organisms also require unique substances such as _________________ used to construct _____________________

silicic acid silica walls of diatoms

What happens to a microorganism if one nutrient is limited or in short supply?

the organism will have limited growth

Decimal reduction time (D value)

time required to kill 90% of the target organism under specific conditions

hydrogen peroxide (H2O2)

toxic oxygen species cellular defense: catalase and peroxidase enzymes

Singlet oxygen: 1O2

toxic oxygen species source: photochemical reaction; product of peroxidase enzymes cellular defenses: antioxidants such as carotenoid pigements

2. Lithotrophs

use inorganic molecules as a source of electrons

What makes a chemical a "good" microbe-killer?

§ Should kill a wide-range of microbes § Shouldn't be corrosive or overly toxic § Shouldn't leave a residue § Shouldn't emit fumes or smell TOO bad § Should be cheap § Should be temperature stable

How do you pick a method for destroying microbes and preventing their growth?

§ What microbes are present? What about endospores? § How many microbes are present? Do they all need to die? (more there are, the longer it will take from them to die) § What kind of object needs to be treated? § If using a physical method, how long/intense does it need to be? § If using a chemical method, how powerful must it be, and how long does it need to be applied? Do we need to worry about toxicity to humans or other life?

Strict Anaerobic Microbes

· All strict anaerobes lack or have low quantities of o Superoxide dismutase o Catalase · These microbes cannot tolerate O2 · Anaerobes must be grown without O2 ---> Gaspak anaerobic system

Medicinal Leech, a Medical Device

· Approved by FDA as a medical device for postoperative relieve of venous congestion o Secrete vasodilators and anticoagulants · In ~20-30% of the patients, Aeromonas can cause wound infections · Ciprofloxacin resistant isolates have been recently detected. · The secretion of anticoagulants and vasodilators and the active removal of blood aid the resolution of venous congestion. · Leeches are not sterile but carry digestive-tract symbionts. · Since the 1980's, reports were published describing Aeromonas hydrophilawound infections in ~5-30% of the patients. · This infection rate dropped due to prophylactic ciprofloxacin treatment.

MacConkey Agar

· Both differential and selective o Contains lactose, dyes (crystal violet and neutral red), and bile salts - selects from gram negative bacteria and for organisms that produce acid from lactose fermentation o E coli is red (ferments lactose to acid) Organism identified: Gram-negative enterics Selective: crystal violet and bile salts inhibit Gram-postive bacteria Differential: Lactose and pH indicator neutral red (red when acidic) identify lactose fermenters as red colonies and non-fermenters as white or tan. most intestinal pathogens are non-fermenters and hence do not produce acid

How do Solutes and Water activity affect microbial growth

· Changes in osmotic concentrations in the environment may affect microbial cells o Hypotonic solution (lower osmotic concentration outside) - Water enters cell - Cell swells and may burst o Hypertonic (higher osmotic concentration outside) - Water leaves cell - Membrane shrinks from cell wall (plasmolysis) may occur · Water activity (aw) o The amount of water that is available to organisms o Reduced by interaction with solute molecules (osmotic effect) --> higher solute --> lower aw o Reduced by absorption to surfaces (matric effect) o aw of distilled water is 1.0 o aw of milk is 0.97 o aw of dried fruits is 0.5 o osmotolerant microbes can grow over wide ranges of water activity (Staphylococcus aureus 3M NaCl --> osmotolerant) o Xerophile - can grow on "dry" surfaces · Effects of osmotic pressure and water availability on microbial growth o Different solute concentrations can result in influx of water into or efflux from the cell - This can cause stress to the cell, causing it to either swell or shrink - Pure water aw= 1; seawater aw= 0.98; honey aw= 0.6 - Most bacteria require an aw> 0.9 - Fungi can tolerate a lower aw

What confers FQ-resistance

· Chromosomal Mutations in the topoisomerase genes - gyrA: primary target - parC: secondary target · Plasmid-associated genes - Efflix pumps (quepAand oqxAB) - Quinolone resistance (qnr) gene family (unknown mechanism) probably bind to FQ

Ciprofloxacin Resistant Genes

· Ciprofloxacin resistance is a multistep process: · In Gram-negative, bacteria typically a mutation in DNA-gyrase, gyrA, occurs first. · Other mutations elevate resistance to intermediate (1 to 4 μg/ml) or resistance ( >4 μg/ml) - DNA topoisomerase IV, parC - Quinolone resistance proteins that bind quinolones, qnr Quinolone efflux pumps,qepA

Microbial Growth on Solid Surfaces

· Colony characteristics that develop when microbes are grown on agar surfaces aid in identification · Differences in growth rate from edges to center due to o Oxygen, nutrients, and toxic products o Cells may be dead in some areas

Tyndallization

· Developed by John Tyndall · Intermittent sterilization · 30-60 min of steam repeated 3 times with 23-24 hr incubations between · Used to kill spores - spores germinate and are then killed by the steam treatment · Useful for soil samples because they have a lot of spores

Viable Counting Methods if microbe cannot be cultured on plate media

· Dilutions are made and added to suitable liquid media · Turbidity determined to yield the most probable number (MPN) o Produces a turbid environment and color change if organism is present o Used for bathing beaches, cosmetics, and food products Con: uses a lot of test tubes

Indirect measurements of cell mass

· Dry weight o Time consuming and not very sensitive · Quantity of a particular cell constituent o Protein, DNA, ATP, or chlorophyll o Useful if amount of substance in each cell is constant · Turbidometric measures (light scattering) o Quick, easy, and sensitive

How does pH affect microbial growth?

· Effects of pH on microbial growth opH affects macromolecule structures and transmembrane electrochemical gradients · Each microbe will have an optimal pH range for growths: Acidophiles, Neutropholes, and Alkophiles

Physical Control Methods

· Filtration · Heat · Radiation

By analyzing the data from a growth curve, one can determine

· Generation time: The time to double the population in the exponential phase · Growth rate: Number of generations/unit of time (inverse of the generation time) · Growth yield: The maximum population density and/or amount of cellular material produced by the culture

Growth of Strict anaerobes in culture

· Growth in anaerobic culture medium containing reducing agents (thioglycollate or cysteine) · Work in anaerobic chamber · Gaspak jar · Plastic bag or pouch system

Extremely adapted Microbes

· Halophiles o Grows optimally in the presence of NaCl or other salts at concentration above 0.2M · Extreme halophiles o Require salt concentrations of 2M and 6.2M o Extremely high concentrations of potassium o Cell wall, proteins, and plasma membrane require high salt to maintain stability and activity (e.g. Halobacterium in Dead Sea)

Extremely adapted Microbes

· Halophiles o Grows optimally in the presence of NaCl or other salts at concentration above 0.2M · Extreme halophiles oRequire salt concentrations of 2M and 6.2M o Extremely high concentrations of potassium o Cell wall, proteins, and plasma membrane require high salt to maintain stability and activity (e.g. Halobacterium in Dead Sea)

Stationary Phase

· In a closed system (i.e., batch culture) population growth eventually ceases, total number of viable cells remains constant (109) o active cells stop reproducing or reproductive rate is balanced by death rate o suspended animation · Reasons for stationary phase o nutrient limitation o limited oxygen availability o toxic waste accumulation o critical population density reached

The Streak Plate (T Streak)

· Involves technique of spreading a mixture of cells on an agar surface so that individual cells are well separated from each other o Involves a bacterial loop · Each cell can reproduce to form a separate colony (visible growth or cluster of microorganisms)

Genome comparisons of leech symbionts - Results

· Isolates from Southern France, Idaho, and California were basically identical · Some clinical isolates from wounds and leech isolates were also identical · A. hydrophila and A. veronii are found in leeches and both can cause infection, but most infections are caused by A. hydrophila

Evaluating a disinfectant: Disk-diffusion method

· Make microbe smear, take disk of disinfectant and lay over top, look at zones of clearing (the larger the zone, the more effective the disinfectant is)

Presence of Plasmid-Encoded Mutations in Ciprofloxacin-Resistant Aeromonas Strains

· Many had FQR and were located on plasmids that were conjugatable, meaning they could be conjugated to other bacteria

How did the Graf Lab's findings on the symbiont of the medicinal leeches differ from other labs?

· Many labs found that Aeromonas hydrophila was the symbiont of the leeches but the Graf lab found Aeromonas verolaii

Presence of Chromosomal Mutations in Ciprofloxacin-Resistant Aeromonas Strains

· Most bacteria had gyrAmutation and all the resistant strains had gyrA and parCmutations · Only two strains that were sensitive did not have the parC mutation

Why is it so difficult to grow Myobacterium leprae in lab?

· Mycobacterium lepraehas a slow growth rate and we cannot figure out how to grow in lab as we do not know growth requirements

Senescence and Death Phase

· Number of viable cells declines exponentially o two alternative hypotheses - cells are Viable But Not Culturable (VBNC) · cells alive, but dormant, capable of new growth when conditions are right - programmed cell death · fraction of the population genetically programmed to die (commit suicide)

How did the lab approach studying Ciprofloxacin resistant infections associated with the medicinal leech?

· Obtained clinical isolates · Cultured strains from leeches on growth media containing Cp · Using biochemical tests, diagnostic PCR and 16S rRNA gene sequencing preliminary identifications were done · Antibiotic resistance profiles were determined

Which organisms were CpR

· One clade of CpR A. veronii and two clades of CpRA. hydrophila

Basis of Different Oxygen Sensitivities

· Oxygen easily reduced to toxic reactive oxygen species (ROS) o Superoxide radical o Hydrogen peroxide o Hydroxyl radical · Aerobes produce protective enzymes o Superoxide dismutase (SOD) o Catalase o Peroxidase

Some Media Components

· Peptones · Extracts · Agar

Phenolics

· Phenol was 1stwidely used antiseptic and disinfectant. Used in 1867 by Joseph Lister · commonly used as laboratory and hospital disinfectants (e.g., Lysol) · act by denaturing proteins and disrupting cell membranes · tuberculocidal, effective in presence of organic material, and long lasting (can kill Myobacterium tuberculosis, which is very difficult to kill) · disagreeable odor and can cause skin irritation · E.g., triclosan is used in hand sanitizers (not allowed in products right now because used so much that bacterial resistance is a fear) (in Colgate toothpaste because it prevents cavities)

exponential population growth

· Population is doubling every generation

Requirements for DNA Sequencing

· Requirements o A molecule that is present in all organisms o Molecule has conserved sites (for PCR amplification) o Molecule has variable sites (for taxonomic information) o The phylogeny of the molecule reflects the phylogeny of the organisms

How to Determine Resistance and the MIC (minimum inhibitory concentration)

· Serial dilutions of antibiotics are performed · Determine the dilution at which growth is inhibited

Growth Factors

· Some organisms can synthesize all organic molecules from a single carbon source and inorganic salts, but some require growth factors to support growth · Growth factors are organic compounds that cannot be synthesized by an organism but are essential for growth · Must be supplied by the environment if the cell is to survive and reproduce

Genome comparisons of leech symbionts - method

· Strains were selected for genome sequencing - Phylogenetic relationship determined (32 Kb from 16 Housekeeping genes) - Can we link clinical isolates to leech isolates? - Can we confirm the species identification? · Determine CpRlevels · Identify mutations or gain of genes associated with CpR

How does temperature affect microbial growth?

· Temperature can affect macromolecule structure, membrane fluidity, and enzyme function · Different microbes have different optimal temperature growth ranges · microbes cannot regulate their internal temperature · enzymes have optimal temperature at which they function optimally · high temperatures may inhibit enzyme functioning and be lethal

Temperature Manipulation

· Temperature manipulation—lower heat o Pasteurization (low-temp heating) to reduce microbe numbers · Temperature manipulation—freezing o Can damage cells by forming ice crystals o Can stop biochemical reactions in microbes o Good for long-term preservation o Will destroy some but not all --> not a good method of sterilization

What functions do symbiotic bacteria perform?

· These bacteria provide important abilities and functions that we cannot do o Nutrients: vitamin K, essential AA o Stimulate immune system o Digest food o Inactivate medications o Protect against pathogens o May prevent onset of certain diseases (IBD, colon cancer, lactose intolerance)

How can we use our knowledge of organism requirements to grow them in the lab?

· To study organisms we need to be able to grow, transport, and store microorganisms in the lab · Culture media is solid or liquid preparation · Culture media must contain all the nutrients required by the organism for growth · The specific requirements of the medium depends on the organism being cultured · Culture media can be classified based on o Chemical constituents from which they are made o Physical nature o Function

Role of Low Antibiotic Concentrations

· Very low levels of antibiotics less than 100 times the MIC are enough to select for resistant strains · Must be careful of contamination to not promote antibiotic bacteria from being successful · Problem because environment has low levels of antibiotics in it

Which strains had CpR

· Wild type + gyrAmutation + parC + pAv42 - (most) - 3 (4 is resistant) · Wild type + gyrAmutation + parC + pEL1 - intermediate (2)

What is needed to grow E. coli that has a mutation in one of the pathways leading to its amino acids?

· Wild type E. coli can synthesize all of its amino acids, nucleic acids, anything it needs given basic requirements for carbon and inorganic salts, but if there is a mutation in one of the pathways leading to its amino acid, this organism would need that amino acid added as a growth factor to growth medium in order for that microorganism to grow

Hirudo verbena

· a freshwater parasite, feeds exclusively on vertebrate blood · Two symbionts dominate crop microbiota o Aeromonas veronii o Rikenella-like bacterium Simple gut community makes this an ideal system to study digestive-tract symbiosis

Exponential Phase

· also called log phase · rate of growth and division is constant and maximal (based on genetics, the medium and environmental conditions) · population is most uniform in terms of chemical and physical properties during this phase (these cells are usually used for biochemical and physiological studies) · during log phase, cells exhibit balanced growth o cellular constituents manufactured at constant rates relative to each other

Halogens

· any of five elements: fluorine, chlorine, bromine, iodine, and astatine · important antimicrobial agents

Nutritional Requirements of microorganisms: Micronutrients

· are also required by microbes o Includes several metal ions (NA+, Mg2+, Mn2+, etc.) o Often required for protein structure/activity, biosynthesis of ATP by electron transport-related processes

Prolonged Decline in Growth

· bacterial population continually evolves · process marked by successive waves of genetically distinct variants · natural selection occurs

Dry Heat Incineration

· bench top incinerators are used to sterilize inoculating loops used in microbiology laboratories

Lag Phase

· cell synthesizing new components o e.g., to replenish spent materials o e.g., to adapt to new medium or other conditions · varies in length o in some cases can be very short or even absent o short if kept in same media as before

Pasteurization

· controlled heating at temperatures well below boiling (55 - 60 C) · used for milk, beer, and other beverages · process does not sterilize but does kill pathogens present and slows spoilage by reducing the total load of organisms present · Introduced to US in 1889 · NOT STERILIZATION · Reduces microbial load and destroys pathogens

Moist Heat

· destroys viruses, fungi, and bacteria · boiling will not destroy spores and does not sterilize · degrades nucleic acids, denatures proteins, and disrupts membranes

Quaternary ammonium compounds

· detergents that have antimicrobial activity and are effective disinfectants o amphipathic organic cleansing agents o act as wetting agents and emulsifiers o Disrupt membranes and denature proteins o kill most bacteria, but not Mycobacterium tuberculosisor endospores o safe and easy to use, but inactivated by hard water and soap o E.g., benzalkonium chloride (food utensils) Sometimes used in hand sanitizers

Dilution rate and microbial growth

· dilution rate - rate at which medium flowsthrough vessel relative to vessel size · note: cell density maintained at wide range of dilution rates and chemostat operates best at low dilution rate. At very low dilution rates energy is limited and cells can't grow - Maintenance energy · high dilution rate will wash cells out

Heavy Metals

· e.g., ions of mercury, silver, arsenic, zinc, and copper (e.g., CuSO4as algaecide in swimming pools, Silver nitrate to prevent ophthalmic gonorrhea - now use erythromycin) · effective but usually toxic · combine with and inactivate proteins; may also precipitate proteins

Stationary Phase and Starvation Response

· entry into stationary phase due to starvation and other stressful conditions activates survival strategy o morphological changes · e.g., endospore formation o decrease in size, protoplast shrinkage, and nucleoid condensation o RpoS protein assists RNA polymerase in transcribing genes for starvation proteins

Ionizing radiation (gamma radiation)

· gamma radiation penetrates deep into objects · destroys bacterial endospores; not always effective against viruses · used for sterilization and pasteurization of antibiotics, hormones, sutures, plastic disposable supplies, and food · Sterilization with Ionizing Radiation

Importance of Continuous Culture Methods

· growth in an open system o continual provision of nutrients o continual removal of wastes · maintains cells in log phase at a constant biomass concentration for extended periods · achieved using a continuous culture system

The Continuous Culture of Microorganisms

· growth in an open system o continual provision of nutrients o continual removal of wastes · maintains cells in log phase at a constant biomass concentration for extended periods · achieved using a continuous culture system

Dry Heat Sterilization

· less effective than moist heat sterilization, requiring higher temperatures and longer exposure times (e.g. incineration or oven) o items subjected to 160-170oC for 2 to 3 hours · oxidizes cell constituents and denatures proteins · E.g., C. botulium- 5 min at 121C or 2 hrs at 160C

Flow Cytometry

· microbial suspension forced through small orifice with a laser light beam · Movement of microbe through orifice impacts electric current that flows through orifice · Instances of disruption of current are counted · Specific antibodies can be used to determine size and internal complexity · Must be clear suspension, and we will not be able to tell if cells are alive or dead

Pattern of Microbial Death

· microorganisms are not killed instantly · population death usually occurs exponentially · measure of agent's killing efficiency o decimal reduction time (D-value) - time to kill 90% of microorganisms and spores in a sample under specific conditions o Z- value- temperature change needed to kill 90% of microorganisms and spores in a sample under specific conditions o must be sure persister cells (viable but nonculturable (VBNC)) are dead --> once they recover they may regain the ability to reproduce and cause infection

Steam Sterilization

· must be carried out above 100oC which requires saturated steam under pressure · carried out using an autoclave (Developed by Chamberland, 1884) (15 lbs/sq. inch) · (121 C at 15 psi for 10-15 min) · effective against all types of microorganisms including spores · quality control - includes strips with Geobacillus stearothermophilus (produces spores - ensures effectiveness of sterilization)

The Growth Curve

· observed when microorganisms are cultivated in batch culture o culture incubated in a closed vessel with a single batch of medium · usually plotted as logarithm of cell number versus time · has four distinct phases o lag, exponential, stationary, senescence (aging), and death o viable cell density is determined by plate count o stationary phase = maximal cell density is reached

Effect of Temperature on Growth Rate

· organisms exhibit distinct cardinal growth temperatures ominimal omaximal ooptimal · membranes melt of solidify · proteins denature

Starvation Responses

· production of starvation proteins - makes cells harder to kill o increase cross-linking in cell wall · Dps protein protects DNA o chaperone proteins prevent protein damage · cells are called persister cells o long-term survival o increased virulence

Adaptations of Thermophiles

· protein structure stabilized by a variety of means o more H bonds o more proline o chaperones · histone-like proteins stabilize DNA · membrane stabilized by a variety of means o more saturates, more branched and higher molecular weight lipids o ether linkages (archaeal membranes)

Unbalanced Growth

· rates of synthesis of cell components vary relative to each other · occurs under a variety of conditions o change in nutrient levels · can respond very quickly to a change in conditions · shift-up (poor medium to rich medium) o more favorable conditions · shift-down (rich medium to poor medium) o less favorable conditions o change in environmental conditions o Demonstrates that microbial growth is under precise, coordinated control and that organisms respond quickly to changes in environmental conditions. Shift-up Shift-down experiment

Biological Safety Cabinet

· surgical masks · N-95 masks removes 95% of particles 0.3um or larger · cotton plugs on culture vessels · high-efficiency particulate air (HEPA) filters o used in laminar flow biological safety cabinets o Removes 99.97% of particles 0.3 um or larger

Sterilizing Gases

· used to sterilize heat-sensitive materials like plastics · microbiocidal and sporicidal · ethylene oxide sterilization is carried out in equipment resembling an autoclave - rapidly penetrates packing materials, even plastic wraps (5-8 hrs at 38C or 3-4 hrs at 54C at 40-50% RH) - very toxic to humans · betapropiolactone and vaporized hydrogen peroxide o combine with and inactivate DNA and proteins · Ethylene Oxide Sterilizer o USED TO STERILIZE PETRI DISHES and medical equipment (no liquid)