unit 2: chap 6 microbial growth
microaerophiles
aerobic conditions but 02 thats less than atmospheric - grow right below top of deep -dont protect themselves ex: C. jejuni, B. burgdorferi, H. pylori
What is generation time in the context of this chapter? What factors could affect generation time?
amount of time needed for a cell to divide and microbial population to double (5 min, 1 day, 2 hrs) factors: dependent on microbe, temp, nutrients
Most Probable Number (MPN)
assess water quality, conc of coloform bacteria in water sample - gram (-), fermenting lactose at 37C w. gas production, no endospores -indicated fecal contamination in water water sample and 2 dilutions, 10-1 and 10-2 -transfer fluid from original dilutions into replicate tubes -replicate tubes have broth, 5 tubes each for 1--1 and 10-2 -incubate, see wether evidence of turbidity, gas production (lactose broths) ,coloform present= gas formation turbidity + gas made= coloform -count positive tubes in replicates yellow = positive 5 positive from set 1, 3 from 2, 1 from 3, check graph to check = 110 cells MPN -statistics using method
What are some examples of beneficial and problematic consequences of biofilm formation?
benefits for microbe: -protected by extracellular matrix (EPS) -biofilm is harder to get rid of - not all microbes need to do every process, perform community tasks
What is the "danger zone" temperature range for food spoilage? This corresponds to the growth range of what category of microbes? At what temperatures should hot and cold foods be kept at?
between 15-50C=danger zone growth range of mesophiles 18C for freezer recommended 0C- for the refrigerator can grow some mesophiles, but not much over 50C= slow growth hot foods should be kept above 140F cold foods should be kept below 40F
What do we typically mean when we refer to bacterial growth?
binary fission - not mitsois, asexual reproduction, does not introduce genetic diversity
Biofilm formation
biofilm deposition of free swimming microbe lands on a solid surface with water - if is able to adhere, it can divide (starts biofilm) -division= produce matrix (EPS), additional microbes can incorporate 1 biofilm- many different genres
What is a biofilm? How are biofilms formed? What is quorum sensing? What is the advantage of a microbe becoming incorporated into a biofilm?
biofilm- aggregate surrounded by extracellualar matrix, adhere inside and outside body outside- access to water -considered hydrogels= water component
quorom sensing
cells of biofilm communicate from Autoinducers - more cells= more autoinducers certain density- autoinducer concentration reached, gene expression changes (matrix production, motility, toxin production) -planktonic needs to move, not important when in a biofilm - wont produce toxin until there is a large number of cells present to persist a host
colonies
circular patterns of growth on solid lab media after incubation
Compare and contrast the different types of media discussed in lecture. What types of substances should chemical media provide to support microbial growth?
culture medium - need moisture, right pH, amount of oxygen, sterile, incubated at the right temperature solid medium: contain thickening agent, most common= agar agar: - most microbes don't make enzymes that can degrade agar, wont liquify 100C, can grow phychrotrophs, mesophiles, (Petri plates, slants/deeps)
Why do we usually plot changes in bacterial populations using logarithmic rather than arithmetic curves?
e. coli gen time= 20 min, pop doubles every 20 min 7 hours= 1 million cells arithmetic= misleading graph, doesn't look like many cells when there actually is logarythmic= generation vs log number, clean line and accurate visual, binary fission= exponential growth time
selective and differential media: EMB
eosin methylene blue (EMB) agar: - selective for enterobacteriacae (E. coli v enterobacter), gram - gut bacteria agent: pigments methylane blue (inhibit gram + bacteria) differential for lactose fermentation: E. coli has enzymes for this fermentation, acid made, colonies to be metallic green enterobacter ferments too, but makes low acid, purple and pink color
chemically defined media
exact chemical composition is known (recipe list) -consistent, same between batches - provides C, N, S, energy, Phosphorous, molecules ex: E. coli - knows exactly what it needs and quantified
Planktonic bacteria
free floating bacteria
mesophiles
grow at intermediate temps range: min: 10C, optimal: 25-40C -live in animals and humans - optimal to hosts temperature -common food spoilage microbes in room temperature -human pathogens and accidentally ingested 2. aureus, S. pyogenes, S. pneumoniae, E. coli, L. monocytogenes (different strains), B. subtilis (soil, GI)
obligate aerobes
grow at the top of a tube -create catalase and superoxide dismutase
biofilms
grow organized in column structures - water can pass through biofilm and contact cells, water provides nutrients - waste products pieces of biofilm break off, spread easily
problems of biofilms for humans
-form on counters, shower, clog drains in body: - form on catheters, pacemakers, joint replacement -patient wont respond to 1000x antibiotic dose, once formed, hard to remove, easier to replace -use antimicrobial chemicals, still hard teeth: cavity forms middle ear infections in children heart valves- bacterial endocarditis
What are some ways in which bacterial cultures can be preserved?
-lower storage temp, longer the cells will keep 1. Refrigeration 2. Deep- freezing - needs cryoprotectant (glycerol), prevents ice crystal formation 3. Lyophilization (freeze-dry) - freezing, then dehydration -powder looking
facultative anaerobes
02- goes to top of the deep, if 02 is gone then use fermentation- goes deeper in the deep -protect from 02 from superoxide dismutase and catalase
What are the chemical requirements for growth?
1. H20 2. Carbon 3. nitrogen- lipids, nucleotides 4. sulfur- AA's 5. phosphorous- ATP 6. K, Mg, Ca, trace elements- cofactors 6. organic growth factors (vit. and AA) 7. Oxygen - some need
Oxygen and microbes
1. Oligate Aerobes: - exposed to oxygen (need it) - use as final e- acceptor -aerobic CR ex: M. pneumoniae, B. pertussis 2. Facultative Anaerobes - use 02 when given, can use fermentation as well when 02 is absent ex- E. coli, S. aureus 3. Obligate Anaerobes - need no 02 -anaerobic only ex. C. botulinum, C. Tetani
What physical growth requirements other than temperature are important in determining microbial growth?
1. pH - narrow range (7) neutral pH 2. Osmotic Pressure - hypertonic= h20 moves out, cytoplasm shrinks, plasmolysis occurs= prevents cells from growing -metabolism stops, cell division stops sugary and salty conditions helps control microbe growth
Describe the streak plate method for obtaining pure cultures.
1. transfer cells from broth onto petri plate using streak plate method - divides plate into 4 sections 1. transfer cells into section 1, flame loop and cool 2. collect cells from section 1 3. drag cells from 2 to section 3, flame loop, cool, repeat 3. streak section 4 from section 3 -reduces number of cells that will be present in each section 3/4 will have cells more spread out, will produce isolated colonie task: make a pure culture - mixture of red colonies and cream colors, red= serretia marcescnens, cream= e. coli transfer whatever cells onto new medium like a slant and incubate, only should see growth of S. marcesscens
estimation of microbial growth: indirect
1. turbidity present - 1 mill cells per mL light rays pass through sample to spectrophotometer, absorbance scale (0 absorbance if no turbidity) when turbidity, not all light rays can pass (most absorbed by sample, but absorbance is high) need to be 10-100 million cells per mL present for broth to be turbid enough to be read 2. metabolic activity
Are antibiotics and other antimicrobials equally effective in treating planktonic and biofilm forms of a microbe?
1x dose for planktonic higher dose by 1000x to impact microbes in a matrix
rice cookers
212F max held at 150F or machine clicks off and held at room temp room temp= DZ -dangerous for elderly or children ex= bacillus serius- toxins, vomiting
anaerobic growth media
harder to grow - media must be deprived of oxygen - use a reducing media (sodium thioglycolate broth)_ reducing, reduces 02 to just h20 visual: methylane blue (blue green with 02) and colorless with no 02 if see growth in green portions, this microbe is aerobic growth if see growth at bottom of broth or clear, indicates anaerobic growth oxyrase also reduces 02 to h20 -chambers with chemical packet soak 02
slow cookers
high setting 250-300F low setting 190-200F both above danger zone warm setting 145-165F - close to DZ
What is an inoculum? A culture? What is a fastidious microorganism?
inoculum- introducing microbes into a culture to promote growth, transfer of cells to sterile culture- (when growing and dividing) microbes that grow and multiply on or in a culture medium fastidious- fussy medium for growth, medium provides what they cant provide themselves
Differential Media
look for blood agar during hemolysis, distinguish between S aureus and E. coli alpha vs beta hemolysis beta hemolysis: break down RBC and hemoglobin= yellow agar alpha hemolysis: partially break down RBC's = biliverdin made (green) green brown gama hemolysis: no hemolysis, no enzymes that break down RBC, agar still red
other indirect estimating means
metabolic activity: measures amt of metabolic product, amt of product measuring is proportional to cells made dry weight: filamentous organisms, microbes dried out, removed from medium, weighed, weight= number of organisms
fungi pH
more acidic - 5.6 pH supports fungi growth -too acidic for most bacteria
complex media
most heterotrophic bacteria and fungi grow here - variation between batches - recipe list known, but variation in ingredients ex: vitamins provided my meat/yeast, if meat varies, causes varyation -proteins provide energy and carbon ex: nutriet agar (solidifying), nutrient broth, blood agar nutrient agar: ingredients like peptones, beef extract, yeast
Obligate anaerboes
no 02, atp in anaerobic only - growth in bottom of deep - dont make protective enzymes SD or C
aerotolerant anaerboes
only anaerobic growth ,can continue with presence of 02 - all throughout the deep - protect by Superoxide Dismutase, and Peroxidase ex- streptococcus, lactobacillus
Distinguish between minimum, optimum, and maximum growth temperatures for each
optimum- denatures past the optimum temperature, max temp they can divide in binarry fission
thermophiles
optimum= 60C dont grow at room temp - produce endospores - survive heat treatment -not a huge health threat -archea: 80-90C (hyperthermophiles) hyperthromophiles- boiling water, volcanoes, make endospores, highly heat resistant, survive for days and weeks
Which is most likely to cause spoilage of refrigerated foods? Which are the most common spoilage and disease organisms?
phychrotrophs= refrigerated foods most common are mesophiles= spoilage and disease
pour plates
pour plate: -small vol of diluted bacteria, add to petri dish, add hot melted agar, solidify, then incubate, then count number of colonies present 1 cell rises to colonies, or a clump of cells gives rise colonie counts= 100 colony forming units advantages: diff microbes can grow. aerobic bacteria on surface, microaerophiles or obligate anaeobes can grow inside agar disadvantage: hot melted agar, too hot= kill cells = no colonies -stacking of colonies, hard to count -accuracy can only happen between 30-300 colonies present, anything else is not gonna give good counts
fastidious microbes
production of fermented foods like sour crout - requires a lot from its media - AA, vitamins, etc that it cant make itself - recipe list clearly identified, and how much to produce the medium
Distinguish between psychrophiles, psychrotrophs, mesophiles, thermophiles, and hyperthermophiles.
psychrophiles- grow in cold temps 1. strict phychrophiles- min= -10C optimal= 10-15C -depths of the ocean, polar area, dont impact food much -enzymes tolerant of cold temps can remain 2. phychotrophs min= 0C optimum= 30C -common in low temp food spoilage in refrigerated food conditions ex: pseudomonas, listeria monocytogenes mesophiles- grow at intermediate temps range: min: 10C, max: 50C thermophiles- grow in hot temps (all limited to narrow range=30C)
selective media
selective agents/characteristics that select growth of one microbe with the expense of another ex: Sabouraud Dextrose Agar- fungi - low pH (5.6), fungi can grow but bacteria will not
selective and differential media: MSA
selective tolerance for salt tolerant gram + bacteria (S. coccus) agent: salt concentration (7.5%) -inhibit gram - bacteria S. aureus, S. epidermidis Mannitol formation: red originally (w/ NaCl, mannitol, pH (phenol red) yellow when acidic (6.8), phenol red when red at alkylane) S. aureus: makes enzymes to ferment mannitol, lot of acid made, turns Phenol Red to yellow S. epidermidis: does not have the enzymes to ferment, does not change (still red)
plate counts with serial dilutions
serial: series of dilutions, link to plate counts, allow to calculate concentration of bacteria in a sample
Is it better to store food in the refrigerator in tall and skinny or wide and shallow containers? Why?
shallow wide container- about 1 hour in danger zone (15-50C) -better to store here tall skinny- 5 1/2 hours in danger zone
spread plate method
solid agar in petri plate, small vol of dilution bacteria and pour on top of agar, spread tool to spread all over surface - few min to soak, then incubate advantage: dont have to use molten agar disadvantage: microaerophiles and obligate anaerobes cannot grow, colonies only grow on the surface of agar,
direct microscopic count
special slide or cell counters - they count both live and dead cells, distinction may be important for what is being determined
requirements for growth
temp, pH, osmotic pressure, chemical
Acidophiles pH
tolerant of acidity, grow in sulfur hot springs or volcanic vents -high temp and acidic medium
enrichment culture
used to make cultures that dont have high representation for initial sample - provide nutrients for further growth, doesnt have inhibitors that promote other growth bacterias - grow select microbes that are in soil or fecal samples, cells collected, transferred to tube w/ same enrichment, several transfers formed
filtration
using for water samples with not large samples of bacteria 100 ml h20 run over bacterial filter, pores in filter allow h20 to pass through but too small to let bacteria leave, bacteria stuck on filter -filter tranferred to growth medium, incubated, 214 CFU's identified 1 cell= 1 colony (not always true) 214 CFU's correspond to 214 cells in 100ml
benefits of biofilms for humans
- roots of plants, helps plants harvest nutrients from soil -remove organic waste from sewage -bioremediation (oil spills)
What is a halophile? Distinguish between obligate and facultative halophiles.
Halophiles - have a NEED for salt or are tolerant to salt, grow in salt enviornments Obligate Halophiles - need salt, thrive in 20-35% NaCl Facultative Halophiles - tolerant, dont have a requirenment - survive NaCl between 2-7% ex: S. aureus
Describe the lag, log, stationary, and death phases of bacterial growth curves
Lag= period of adaptation to medium, not cell growth, no binary fission, need time to adapt, increasing metabolic activity (synthesizing enzymes), depends on: similarities of mediums, temp, # of cells, time to make enzymes log= exponential growth period -binary fission, generation time, doubling in division, predictable intervals, metabolic activity (most abundant here), many nutrients available -gives BEST GRAM STAIN RESULTS Stationary= equallibrium cells dying balanced by cells that are rising - nutrients limited, many cells, not enough, competition, waste products growing, pH decreasing, o2 limited Death= period of death - no cell division, cells dying, reverse of log, this is a period of exponential death -nutrients limited, many waste buildups, impacts cells, continues until population dies out
Identify toxic forms of oxygen. How do bacteria protect themselves from toxic forms of oxygen? Do all bacteria have these protective features?
Reactive Oxygen Species: - 02 aerobes need to peorect themselves 1. singlet oxygen 2. superoxide radicals - o2 aerobes create enzyme Auperoxide Dismutase to help protect 3. Hydrogen peroxidase - 02 aerobes use catalase and peroxidase 4. Hydroxyl radicals - OH
