MIcroBiologyLabs 17-28 Exam Flashcards
(99 cards)
1
Q
Why is milk biologically important?
A
- Milk is a “rich medium”
- -Contains sugars and other carbohydrates, animo acids and proteins, fats, vitamins and minerals
- Supports growth of mammals and bacteria (cause of spoilage)
2
Q
What are the three most common pathogens and their diseases found in milk?
A
- Salmonella: can cause intestinal infection
- Listeria monocytogenes: cause high mortality rate infections of the blood and CNS
- Mycobacterium tuberculosis: causative agent of the lung disease, tuberculosis
3
Q
Pasteurization
LTLT
HTST
A
LTLT: 63C (145F) for 30 mins
HTST: 72C (161F) for 15-20 seconds
*notice that an increase in temperature of about 10C results in a reduction in time for pasteurization of ~50-fold
4
Q
Does pasteurization sterilize milk?
A
- Pasteurization reduces microbial populations by 10,000 -100,000 fold but LTLT & HTST do not sterilize the milk.
- It takes time for populations to recover to the point of spoilage by lactic acid/ other acids through ferm.
5
Q
Two benefits of pasteurization
A
- Eliminates all or almost all pathogen cells, rendering milk safe for human consumption
- Significantly increases “shelf life” or the time until the milk is spoiled by Bacillus and by lactic acid bacteria species.
6
Q
Semi-log paper
plotting & reading
A
- Uses a log/exponential scale on Y-axis, each major division is marked as “1__” or “10” represents a one log unit or 10-fold change between major units. Y-axis should be marked with whole log units/powers of 10 that correspond to your data
- Top Y-value’s major division should be higher than you CFU/ml count. then number down. Then label minor divisions (do not correspond to 1-10)
7
Q
Decimal reduction time
A
-DRT: time needed to reduce microbial pop to a density that is one decimal point to the left of starting density, one power of ten/ one exponent unit/ one log unit less than the starting density/ 90% less than starting density–same as reducing pop to 10% of its original size
Ex:
-1000.0 to 100.00
-(1X 10^3) or (LOG10=3) to (1X 10^2) or (LOG10=2)
-90% 1000 to 100; 100 is 10% of 1000
8
Q
DRT
-Calculate
A
- Plot, draw straight line
- Select two major devisions on Y-axis (1X10^3, 1X10^2. etc) that are both crossed by the curve and are one log unit spare 910-fold)
- draw horizontal line from both to the line and then draw vertical lines down to X-axis.
- Subtract the time point top value from bottom = this is equivalent to time required to reduce the pop from 1X10^(X+1) to 1X10^X (time to reduce pop by one log unit/one power of ten)
9
Q
DRT
-Know how to use DRT to calc time to one CFU/mL
A
-Total time needed to reduce a population from an initial density to a density of one cell/ml is:
LOG10 of initial cell count X DRT
Ex: LOG10 of 5000 X 60 sec (per log unit reduction)= 3.7 X 60 sec = 222 sec
10
Q
Probiotic species
How do they protect us against disease?
A
Bacteria (also called normal flora) that colonize in the large intestines and protects us against disease by manufacturing vitamins that can be absorbed into the blood stream and take up space–inhibiting other pathogenic species by out competing for space & nutrients.
- Thus preventing pathogens from infecting and causing disease.
- Species include:
- Lactobacillus acidophilus
- Lactic acid bacteria
- Bifidobacterium animalis
11
Q
What are lactic acid bacteria?
A
- Gram positive
- Named for end-product of fermentation pathways, important in cheese and yogurt making
- Collection of species in a genera
- Lactococcus
- Lactobacillus
- Leuconostoc
- Streptococcus
12
Q
Bacterial species used to make yogurt from milk
A
- Lactobacillus bulgaricus
- Streptococcus thermophilus
- Produce large quantities of lactic acid by fermentation–which is anti-microbial preservative & accounts for the flavor of dairy product.
- Both are thermophilic and can grow well at temps up to 55C–killing unwanted bacteria & encourages rapid lactic acid production
- Acid will continue to inhibit other spoilage microbes after yogurt is cooled
13
Q
Why is it useful to be able to isolate and identify bacteria?
A
- Identifying and treating human infection
- Revealing contamination of food and water
- Understanding the role of bacteria in ecosystems
- Discovering commercially useful strains of bacteria
14
Q
Selective Media
A
- Selects for and favors the growth , “desired”, microbial groups or species of interest
- Prevents, inhibits or reduces the growth of interfering or undesired species
15
Q
Differential Media
A
- Aid in identification of various microorganisms
- Usually is the presence of specific metabolic substrates, chemical dyes and pH indicators that reveal cell’s ability to preform a particular rxn.
16
Q
Biochemical Fingerprint
A
- Test for a series of “plus” “minus” results to assays which identify if a particular substrate was used and/or a product was produced
- Minus: enzyme was absent–nothing happened
- Based on the presence or absence of enzymes and/or the ability or inability to use certain substrates and produce certain products–these results are compared to known patterns of bacterial species
17
Q
Characteristics of genus Bacillus
A
- Gram positive rods
- aerobic or facultative bacterial species usually found in soil and water.
- Most do not cause human disease w/ exception to B. anthraces (anthrax) and B. cereus (food poisoning)
- Produce endospores which can withstand 70-80 C for 15-20 mins.
- Most Bacillus species are mesophilic but b/c spores form species can be thermoduric
18
Q
Selecting and Isolating cells form genus Bacillus.
What traits make this possible?
What media do we use and why?
A
- Can withstand higher temps–> 70-80C for 15-20 mins
- Secrete extracellular enzymes that are secreted in soil and take advantage of the carbon and energy sources–converting large unabsorbable macromolecules into smaller, adsorbable compounds like sugar and amino acids.
- Extracellular amylase and protease-secreting Bacillus species can be differentiated from cells that lack them through use of media that contains starch and proteins.
- Starch agar & the starch hydrolysis or amylase assay
- Skim milk agar and the casein (protein) hydrolysis assay
19
Q
Know the common traits
Streptococcus and Enterococcus share
A
- Gram positive cocci
- Long chains (strepto) of up to 30 cells b/c cells divide in one plane and remain attached after division
- Do not produce endospores
- Aerotolerant–do not use O2, rely solely on fermentation producing lactic acid. Grow better reduced O2 and elevated CO2
- Catalase negative (typical for cells that do not use oxygen)
20
Q
How do we distinguish Strepto/ Entero from Staphylo/Micro?
A
- Gram stain~ Strept/Ent chians while Staphylo/Micro are cluster/tetrad
- Catalase assay: Strept/Entero are catalase negative while Staphylo/Micro are catalase positive
21
Q
Describe the Catalase assay
Know basic principles
Rxn involved
What is pos & neg rxn
A
-Detects presence of specific enzyme, catalase, which is a O2 handling/ oxygen-detoxifying enzyme. This enzyme is essential to cells that carry out aerobic respiration–mostly found in strict aerobes and facultative anaerobes.
-Catalase eliminates reactive H2O2, a byproduct of O2, by converting it to H2O and O2
-POS rxn: When hydrogen peroxide is exposed to catalase gas bubbles are produced (foaming)–Staphylococcus or Micrococcus
NEG rxn: no bubbles in peroxide solution can be Streptococcus of Enterococcus
22
Q
Is blood agar selective, differential or both… why?
A
-When Streptococcus species is suspected blood agar is used b/c it is a nutrient rich medium composed of peptones, yeast extract and liver or heart extracts with 5% sheep’s blood added to the medium after autoclaving.
-Medium is opaque or cloudy due to presence of intact RBC
-Not very selective b/c nutrients can support growth of a wide range of organisms
It is a differential medium b/c bacterial species break apart RBC (or don’t) differently
-Breaking RBC called “hemolysis”
23
Q
Know & describe the lysis seen in:
beta hemolysis
alpha hemolysis
gamma hemolysis
A
- Beta hemolysis: “bad” secrete toxin polypeptides called streptolyins–will lyse RBC completely. Produces a clear, transparent slightly yellow tinted zone of clearing or hemolysis around colonies
- Alpha hemolysis: Also called green hemolysis, damages but does not completely lyse all the RBC–stays cloudy from remaining RBC–there is a distinct color change in the medium due to conversion of red hemoglobin to a greanish brown pigment biliveridin~partial breakdown damage of tissue
- Gamma hemolysis: Do not lyse RBC–no hemolysis No clear zones appear–medium remains bright red & opaque
24
Q
What are the “virdans” streps?
A
- “normal oral strepts” usually alpha hemolytic
- S. mutans~converts sucrose into gummy plaque and secretes lactic acid causing tooth decay
25
What is the common alpha hemolytic pathogen?
- Strept pneumoniea:
- Cell are arranged in pairs called "diplococci" and can be recognized by shiny mucoid appearance (a product of protein polysaccharide capsule around the cells)
- Unlike viridans species--it is sensitive to anti-bacterial chemical called optochin (ethylhydrocupreine hydrochloride) can be seen by placing "p" disk on blood agar
26
Common beta hemolytic pathogen
- Strept pyogenes: causative agent of pharyngitis--strept throat. "pyo-" ,meaning "pus" which can be seen in throat and tonsils. Can occasionally lead to scarlet fever and rheumatic fever, and life threatening blood infection.
- Strept agalactiae: may be part of "normal flora" of digestive tract/ vagina--usually does not cause problems but can cause serious infection in newborns or neonates following vaginal birth. babies may develop septicemia, pneumonia or meningitis. Also common cause of mastitis (inflammation of cow udder)
27
Common gamma hemolytic pathogen
- Entero Faecalis: A large intestine/ fecal enterococcus species
- usually gamma but sometimes alpha. their presence in water usually indicates fecal-contamination
- Usually there is no damage but cells can relocate and cause UTI like cystitis & if introduced into blood by drama that ruptures large intestines--cells can colonize the heart valves and cause endocarditis
28
What are Lancefield groups?
-A further sub-divided system for Streptococcus based on unique 3-D shape and these unique shapes can act as "antigens"~ surface markers
"Group A" strains carried Group A antigens etc all the way to Group U
- Mostly for dividing beta hemolytic species, alpha and gamma Strept species lacked the carb antigen used in this system--so there is no Lancefield group designation for species such as Strpt pneumoniae & for "virdans" groups.
29
What molecule (protein, lipid or carb) is used as the marker for Lancefield groups?
- Carbs are used as markers--antigens.
30
Know table on p. 229
| summary
- Viridans--strept species produce Alpha(green-brown) hemolysis--no lancefield antigens--is resistant to optochin, usually found in the mouth & upper respiratory--normal oral flora rarely causes disease except tooth decay by S. mutans
- Strept pneumoniae produces Alph (green-brown) hemolysis, no lancefield antigens--is SENSITIVE to optochin, found in upper respiratory tract--common cause of pneumonia, meningitis "pneumococcal diseases)
31
Know table on p. 229
summary
cont.
- Strept pyogenes--Beta (clear) hemolysis, Group A Lancefield group is resistant to Optochin, found in the upper respiratory tract, causes strept throat, scarlet fever, rheumatic fever, pneumonia, septicemia
- Strept agalactiae--Beta(clear), Lancefield group B, Resistant to Optochin, usually found in large intestine/vagina--causes neonatal infections including septicemia, meningitis, and pneumonia
32
Know table on p. 229
summary
cont.
Entero fawcalis--Gamma(red) or Alpha hemolysis, Lancefield group D, resistant to Optochin, normally found in large intestines--normal intestinal flora, usually harmless, may cause urinary tract infections, endocarditis
33
Common traits of Staphylococcus & Micrococcus
- Gram positive coci
- micrococcus--tetrads
- staphylo-clusters
- Do not produce endospores
- Adapted to salty 7.5%NaCl...found on human body
- Uses O2 in aerobic resp.
- Micrococcus--strict aerobes and Staphylo are facultative
- Catalase positive good for distinguishing form other gram pos
34
Four main Stapylo/ Micro species...what do they look like & are they disease causing?
- S. aureus: serious skin infection including boils and carbuncle, wound infections, septicemia and pneumonia. May also cause food poisoning. MRSA
- S. epidermidis: not considered pathogenic rarely cause disease except when introduced into the body via catheters or other med devices--can cause infection of heart valves
- S. saprophyticus: can cause UTI otherwise not a great threat
- M. luteus: found on skin and soil--may rarely cause opportunistic blood infections in immuno-suppresed patient
35
How do we distinguish Staph/Micro form Strept/Entero?
- Gram stain (clusters/tetrads) verse (chains) respectively
- Catalase assay (pos) vs. (neg)
- Salt tolerance (Staphylo and Micro will grow)
36
| How do we distinguish Staphylo from Micro? Assay?
Phenol Red Carb Assay is used to asses a given microbes ability to produce intracellular enzymes needed for fermentation.
pos for acid by fermentation: broth will be bright yellow indicating Staphylo species
neg for acis by ferm: any orange or red color indicating Micrococcus
37
How do we identify species w/in Staphylo genus?
- Fermentaion of mannitol
- resistance to antibiotic novobiocin
- production of coagulase
38
What is Mannitol Salt Agar?
Is it selective, differential or both?
What are the detecting medium... and pos/neg rxn
-highly selective for gram pos ^^
also differential b/c sugar mannitol and pH indicator phenol red--test for ability to ferment mannitol helps to identify staphylococcus species
Pos for acid ferm: if medium around colonies has a bright yellow color could be S. aureus or S.saprophytuicus
Neg for acid ferm: medium will be orange-red to pink red color. this could be S. epidermidis or any number of Micrococcus
39
What is novobiocin? Which species are sensitive to it? Which specie(s) are resistant? How can you tell?
S. saprophyticus is resistant while micro-and other staphylo are sensitive
zone of inhibition 18mm or 9mm radius
40
What is the coagulase enzyme?
| Why is it beneficial for bacteria to make coagulase?
- Coagulase enzyme is a surface protein/ firinogen receptor/clotting factor in Staph aureus
- Benefits:
- Fibrinogen is a plasma protein that normally coats cells and implanted devices--when S. aureus cell interact with fibrinogen it sticks and colonizes
- Coagulase catalyzes the formation of fibrin--fibrin coats the bacteria and makes cells more resistant to microbe-destroying phagocytic WBC
41
| How do we detect coagulase? What do the pos/neg rxn look like?
Use color latex beads coated in fibrinogen--if coagulase is present it will interact with the fibrinogen and link the beads together
-Positive rxn: appears blue grains suspended in colorless liquid within 30-60 sec can be identified as S. aureus Neg rxn: absence of blue aggregates and retention of homogenous opaque blue appearance after 30-60 sec
42
Why must we read coagulase assays sooner rather than later?
False pos possible if wait too long
43
It could be beneficial to be familiar with flowchart on p242
-Catalase:
-Neg: Strept Enterococcus
-Positive:
Glucose Fermentation:
-Neg: M. luteus
-Pos:
Mannitol Fermentation:
-Neg: Staph epidermidis
-Pos:
Novobiocin resistance and Coagulase:
-Neg Coagulase/Novo R: Staphy saprophyticus
-Pos Coagulase/Novo S: Staph aureus
44
Know the common traits of the genus Pseudomonas.
| Where are species usually found?
- gram neg rods
- do not produce endospores
- possess flagella and are motile
- many produce blue, hello, green and or red pigments which are water soluble and diffuse into bacteriological media
- strictly respiratory--relies on ETS NO ferm pathways
- Some are strict aerobes
- others can live anaerobically if nitrate is available
45
Why is Pseudomonas aeruginosa clinically significant?
- Rarely causes human infections 1 exception: Pseudomonas aeruginosa
- Free living--found in soil and water but can also infect plants, humans and other animals.
- "opportunistic" infections tend to be in weaker, injured or vulnerable by preexisting condition or declined immune system
- Burn infections, pneumonia & other lung infections in patients with cystic fibrosis
- Septicemia this species is already resistant to many anti-biotics
46
How do you distinguish Pseudomonas aeruginosa from other gram negative rods?
- Strict aerobes including Alcaligenes, Bordetella and Campylobacter species
- Fermenting facultaive anaerobes like the Enterobacteriaceae or "enteric Family" a group encompassing several genera of gut-adapted bacteria. BUT Pseudomonos aeruginosa & othr Pseu specified differ in that they oxidatively produce acids from glucose
47
What are the three possible results of the O/F assay?
| What organism does each possibility indicate?
glucose medium designed to detect conversion of glucose to organic acids either oxidatively or fermentatively
At pH 6.0--uncharged bromthmol blue molecules outnumber neg charged--clearly yellow--significant quantities of organic acids are produced from glucose--medium will be yellow
6.5-7.5pH 7pH neutral even color molecules will appear green
8.0pH shortage of free protons or H+ neg chraged blue alkaline
open tube yellow at top green at bottom and closed tube green throughout: Ps. aeruginosa
- opentube yellow from t to b and closed tube is yellow from t to b: enterobacteriaceae family
- open tube green to blue at top and green at botom and closed tube is gren from t to b: can be a diff pseu species or enterio
48
Characteristics of Enteric species
- Gram neg rods
- Do not produce spores
- Species are all faculatitive anaerobes produce organic acids and gases
- All enterics ferment glucose to produce organic acids and gas
49
What enzyme must be present for bacteria to be able to use lactose?
What do we call Lactose fermenting species?
What do we call non-lactose fermenting species?
- Beta galactosidase enzyme must be present to split the disaccharide lactose into the monosaccharides glucose and galactose--then cells produce enzymes to convert galactose into glycolysis intermediates
- Able to ferm lactose: coliform
- Not able to ferm lactose: non-coliform
50
Which species are Coliform?
| Which are non-coliform?
Coliform: E.coli, Enterobacter, Klebsiella and many stains/species in Citrobacter genus
Non-coliform: almost all species and strains in genera Proteus, Salmonella, Shigella, Serratia, and Providencia
51
What ingredients in MacConkey agar make it selective?
| Which make it differential?
- Selective for gram neg enterics by inhibiting gram pos with crystal violet and bile salts
- Differential seperates enteric isolates into lactose fermentor and non fermentors. contins 1% lactose and neutral red as pH indicator
52
What does a positive rxn for lactose fermentation look like on MacConkey agar? & Neg rxn
- Pos rxn: medium surounding colonies and colonies are bright red = coliform ~ E.coil and Citrobacter freundii
- Neg rxn: remains unchanged or becomes pale yellow as aposed to brick red
53
What ingredients make EMB agar selective & which make it differential?
- Selective: inhibits gram pos with dyes eosin and methylene blue
- Differential 1% lactose as substrate for ferm. dyes act as pH indicators
54
What does a Pos/Neg rxn for lactose fermentation look like on EMB agar?
& especially high amounts of acid?
Pos rxn: medium surrounding colonies/conolies are dark red-purple to purple black = coliform... high concentrations will produce metalic green sheen typical of E.coli
Neg rxn: remains colorless or yellow purple noncoliform
55
Phenol Red Carb Assay
detects fermentation of sugars to a variety of O-acids and gas end products
Contains peptone and beef extracts & 0.5-1% sugar
```
Pos for organic acid: Tweety bird yellow <6pH
Neg for organic acid: orange to red
Yellow=uncharged (acidic)
Red= become - charged/loss of H+ (basic)
Pos for gas: bubble in durham tube
Neg: no bubble
```
56
Lysine (amino acid) Decarboxylase
Under acidic & anaerobic conditions, will remove the carboxyl (-COOH) group from lysine producing CO2 and an amine called "cadaverine" --reveals rise in pH due to accumulation of alkaline amine in the medium
Pos: pH well above 7.0 light to dark purple
Neg: pH below 6 yellow to dark yellow
57
Methyl Red Assay distinguishes what?
Why is it useful?
MRVP-
-Detects the range of acid produced
-Useful in distinguishing w/in coliform species based on quant it of certain end-products
MRVP (Methyl Red Voges-Proskauer) assay
58
Components of Methyl Red broth
| & what the ingredients do
- Peptones provide C, N, and Glucose act as substrates for ferm & C/energy source
- Buffer: Dipotassium phosphate
- Methyl red is added after inoculation & incubation
59
Pos/Neg rxn for MRVP broth?
In what context of seperating coliform species, why would we want to distinguish between E.coli & E. aerogenes?
Which corresponds to rxn outcomes
- Pos for high conc of organic acids: orange to red indicator only turns orange elow 5PH indicates cells must be E.coli
- Neg rxn: yellow could be E. aerogenes
60
| Citrate assay detects-- Know the components and their purpose
-Simmons citrate agar diffierentiate Ecoli (citrate neg) from E. aerogenes (citrate pos)
To determine if a cell can use citrate as the sole C source.
ammonium is the sole source of N
Bromthymol blue is a pH indicator (also used in O/F assay)
61
Citrate assay rxn outcomes
| The accumulation/lack of what compounds cause these changes?
Pos rxn:visable evidence of growth is a deep or royal blue color indicates ability to use citrate as the sole carbon source must be E. aerogenes
Neg rxn: no evidence of growth medium is green must be e.coli and are likely of fecal origin
a heavy innoculated agar might appear slimy and produce false pos
62
What does Urease assay detect?
| What species is most closely related to a pos urease assay?
- Detects ability to produce an enzyme "urease" whihc breaks down urea into ammonia and CO2
- Proteus species can quickly generate large amounts of urease esp in broth medium.
63
Components of urea broth & purpose of ingredients
- 0.2% urea as substrate for urease activity
- Yeast extract added to provide C, N, and vitamins
- Buffer: mono/di-potassium Phosphate
- pH detector: Phenol red (but looking for raise in pH)
64
Urease rxn outcomes:
Pos: if broth turns bright pink (ammonia was produced) most likely proteus
Neg:unchanged or orange in color no more than 24hrs after in.
65
Three traits SIM assay tests for
sulfide production
indole
motility
66
Know the rxn that occurs in sulfide production.
| How do we detect production of H2S?
- Designed to detect production of H2S from inorganic salts containing sulfates or thiosulfates/ sulfur containing amino acids such as cysteine. all are provided in medium by ferrous ammonium sulfate, sodium thiosulfate and amino acids in peptones.
- Sulfide pro from inorganic salts+ reduced sulfur from +6(sulfate) or +2(thiosulfate) to -2(sulfide) oxidation state
- From cysteine--removal of sulfhydryl(-SH group) from amino acid producing H2S & amino acid alanine
- Smells and black(contains ferrous sulfides
67
What does a pos/neg rxn for sulfide production look like?
sulfide:
pos-black precipitate could be salmonella, proteus or citrobacter
neg- no black precipitate could be several different bacteria
68
Know the rxn for indole production.
| How do we detect the production of indole (reagent) & what is the active ingredient in the reagent (how does it react)
Detects the breakdown of amino acid typtophan to indole, pyruvate and ammonia
- Active ingredient: Kovac's reagent (an organic solvent, butanol.
- Indole dissolves in organic solvents--so indole should be extracted by butanol
- Then indole reacts with Kovac's via aldehyde group to produce cherry red di-dimethyl ammonium salt
69
Indole assay pos/neg rxn outcomes
- Pos if butanol react and produce cherry red could be E.coli and E. aerogenes (have genes for enzyme tryptophanase)
- Neg- yellow or unchaged could be E.aerogene and klebsiella pneumonia
70
Know the term "Peritrichous flagella"
- Most enteric species have "peritrichous flagella"
- Cells have multipe flagella distributed uniformly over the surface of the cells
- 30-40nm in diameter--too thin to be seen with light microscope
71
How does motility assay work? (describe how you preform the assay and why each step is done)
- Detects presence of flagella based on structures ability to move the cell.
- Cells are deposited in tube agar in a neat straight line w/ needle.
- 0.35% agar (semi-solid agar)
- As motile disperse/grow/divide accumulate where medium is cloudy/turbid w/c cells.
72
| What does a pos/neg rxn for motility look like?
pos: disperase of cells, includes all enterics except klebsiella and shigella
neg: still see stab--cells were confined, the rest of the medium should remain transparent, can be Klebsiella or shigella
73
Bacterial Strain
-Derived from single cell, genetically identical
74
Bacteria Species
-Collection of strains
75
Biocode
-The number is a product of all the results from all the assays--it can be matched to a specific species of enteric using Enterotube Interpretation Guide
76
What are the three things we need to know in order to identify a large number of unknown cultures?
1. need to inoculate many different biochemical assays in a short period of time
2. Need to know about variation in biochemical or metabolic activity--construct data base of variation
3. identification system that includes atypical results
77
What is the Enterotube II system? How does it work? How does it account for variations within species?
-A rapid multitest system used to identify gram neg rod species in the family Enterobacteriaceae (enterics)
78
How many chambers do you innoculate? How many assays can one Enterotube preform?
- 12 chambers with different media for biochemical tests
- Use needle at one end of the tube from an isolated colony--inoculating tube at one time
- Total of 15 Assays preformed with 12 mediums
79
Know how to "score" an Enterotube and how to obtain a biocode
- Positive rxn--Circle number
| - add each section til 5 # = code
80
Why don't we test for pathogens directly in water? Know the four reasons
1. Pathogenic cells & virus particles are usually present in [low]
2. Pathogenic bacteria prob won't multiply in H2O so # remain low
3. Many types of pathogens esp viruses are difficult to isolate & culture
4. Many types of pathogens--testing for all would be expensive & time consuming
81
What do we look for instead? Why? What makes them good indicators?
-Coliforms
- Coliform sub-group of enterics can rapidly produce produce acid & gas from fermentation of sugar lactose which will be used to detect fecal contaminated water.
Non-pathogenic usually
82
Know the four reasons listed
1. easy to detect and quantify
2. Usually rare or absent in uncontaminated water
3. Almost always present if intestinal pathogens are present
4. population densities are usually related to degree of fecal contamination
83
What are the three steps/tests we do to analyze water for the presence of fecal contamination? Describe steps in detail
-Presumptive: Uses lactose substrate and durham tube in broth to detect gas/ acid production from lactose ferm
-Confirmed: EMB plate is inoculated with pos from prior test. Should have green sheen for Ecoli or dark purple for E.aerogenes/ other lactose fermentors
Completed: inoculated lactose broth from colony from pos EMB plate & nutrient agar slant. Incubate then if there is a bubble and after a gram stain that indicates gram neg rod
84
What are the two common types of pathogenic enterics in meat? What makes these bacteria pathogenic?
- E.coli-- O157:H7
| - Salmonella--S.typhi
85
What is Hektoen Enteric (HE) agar mainly used to identify? What components of HE agar make it selective/differential?
Selective: for gram neg inhibit gram pos from bile salt and dyes thymol blue and acid fuchsin.
Differential:check for sugar fermentation lactose or sucrose fermentors and nonfermentors
pathogenic enterics do not ferment either lactose or sucrose.
HE contains 1.2%lactose and 1.2%sucrose.
86
| Pos/Neg rxn for lactose(or sucrose) fermentation look like on HE. What bacteria corresponds with each rxn outcome?
Pos rxn: if colonies and the medium surrounding are pink-orange or salmon in color w/ or w/out hazy bile salt precipitate--ferments lactose and maybe sucrose too for organic acids--coliforms probably NOT salmonella or shigella & belong to coliform species or genus like E.coli, enterobacter or citrobacter
Neg rxn: green/blue no hazy bile salts probably is salmonella, shigella or proteus (noncoliform)
87
What does a pos/neg rxn for H2S look like?
Hydrogen sulfide production
Pos rxn: if colonies have black center--salmonella
Neg rxn: colonies do not have black center--Shigella.
88
What would a Salmonella colony look like on HE agar?
| What about Shigella colony?
- Salmonella & Shigella would be green to green blue in color & no hazy bile salt precipitate (does NOT ferment lactose or sucrose to O-acid)
- Salmonella for H2S: positive produce black dot in center
- Shigella for H2S: neg-no h2S production
89
What is nitrogen fixation.
| What are two kinds of nitrogen fixation?
- Reduction of atmospheric N (N2) to ammonia or ammonium (NH3 or NH4)
- Symbiotic nitrogen fixation
- Non-symbiotic nitrogen fixation
90
Describe symbiotic nitrogen fixation.
What types of bacteria do this?
How is the relationship between plant and bacteria symbiotic?
- Bacterial species Rhizobium. The bacteria invade plant roots o the "legume family" & establish symbiosis
- Rhizobium cells enter the plant roots, plant forms protective nodule around bacteria and supplies them with energy.
- In exchange bacteria fix N using nitogenase(enzyme). Produces ammonium which is used by bactera for proteins, nucleic acids, growth and cell division.
- Excess ammonium is diffused out of cells and is absorbed by legumes. ~so in exchange for protection and sugars--the plant gets built in fertilizer
91
How can we observe nitrogen-fixing bacteria? What do they look like once stained?
- "Infected" plants with Rhizobium will have nodules on the roots that are visable. If nodules is crushed on a slide--bacteria can be seen.
- Before joining symbiotiosis with roots--Rhizobium are clearly gram neg rods but once symbiosis is established--they are more Y or club shaped due to plant derived membrane~ bacteroid forms
- Contains inclusions or granules composed of hydroxyl-butarate (small lipid) which usually stain gram pos.
92
Describe non-symbiotic nitrogen fixation. What types of bacteria do this?
- Bacteria capable for fixing N without symbiosis--they are "free-living"
- Cyanobacteria live in water and moist soil. They are able to fix N from N2 gas and photosynthesis--using light energy to create glucose by fixing CO2 gas
- Azotobacter soil dwellinh bactera can't photosynthesize and needs organic molecules for energy and C source.
93
How do Mannitol Salt broths select for nitrogen fixers?
What species does the medium specifically select for?
What do these cells like like under microscope?
- Mannitol salt broth selects for N-fixers by inhibiting non-N-fixers by not providing any N sources in medium. N-fixers will use N2 from the air. Medium does provide a variety of salts for P, S, K and C needs.
- Azotobacter cells can survive on mannitol as sole carbon and energy source --other N-fixers need other organicn molecules (sugars/amino acids from peptones) for growth
- Appear as ovoid "cysts"
94
What are the three steps for nitrogen fixation?
-
95
Describe ammonification,
What is released?
How do we detect it?
What colors indicate a high concentration of the product?
- Release of NH3(ammonia) and NH4(ammonium) from organic N sources such as AA in which N is present as amino (-NH2) groups. Part of decomp of dead plants & animals.
- Released NH3/NH4 can be absorbed by microbs/plants and reassimulate into AA and proteins for themselves.
- Ammonium can also be used at energy source by nitrifying bacteria
- Detection of ammonia/ammonium release by 4% peptone broth (providing a lot of -NH2 rich) amino acids
- Ammonia is detected using ammonia test strip w/ two pads--top has a base that will raise pH of liquid to >pH 10 inwhich ammonium(NH4) is convereted to ammonia (NH3). Second pad contains indicators & dyes
- Color changes from yellow/green to green to green-blue as [ammonia] increases
96
Describe nitrification.
Where can we find bacteria that do this?
Be sure to know the difference between
Nitrosomonas and Nitrobacter
- Oxidizes ammonium (NH4) to nitrite (NO2-) & then oxidizes nitrite to nitrate (NO3-) all under aerobic conditions
- Nitrosomas then Nitrobacter--both are autotrophic (can make sugars and all organic molecules from CO2) but are also chemotrophic (not photosyntetic, must oxidize reduced molecules to acquire energy needed for C-fixing (CO2 into sugar)
- Oxidation of reduced N in forms of ammonium (Nitrosomas) and nitrite (Nitrobacter) that provides energy for C-fixation
- Changes from (+ charged) NH4 to (- charged) NO3- soil particles are net neg and thus nitrate leaching occurs b/c (- &-) repel
97
How do we detect nitrification?
| Describe the components of the media and the components of the test strips used
- Detect nitrification with test strips that both contain sulfanilic acid which react with nitrites to form diasonium salt--whihc reacts with indicator producing pink to red color--red color deepens with increasing [nitrite]. Bottom pad additional secret ingedient that will reduce any nitrates in water to nitrites and produce pink to red color--measuring both nitrate & nitrite in broth.
- Media contains CaCO3 for CO2 and (NH4)2SO4 for added ammonium needed by Nitrosomonas as energy and N source. Once nitrites are produced Nitrobacter can also use nitrites as energy source.
98
Describe denitrification.
What bacteria are involved in this process?
How does this process affect farmers?
- Denitrification/ dissimilatory nitrate reduction use nitrate (NO3-) in place of O2 when bacteria in the genus Bacillus and Pseudomonas are in an anaerobic environment. (Wetlands/flooded ag field) Nitrates are used as teh terminal elector acceptor.
- As a result (depending on pH) the N in NO3- will be reduced to either N2 gas or nitrous oxide (N2O)
- Farmers add nitrogen to fields and if anaerobic conditions persist then the expensive fertilizer is lost into the air.
99
How do we detect denitrification?
Why can we only say "denitrification could have occured"?
What measures do we take to reduce the risk of this false positive occuring?
- Detect denitrification by production of bubble in Durham tube.
- Bubble might be from fermentation of sugar--by chemoheterophs--not from N2 or N2O being released. But to decrease risk of false positive-- medium contains no added sugars, but trace amounts could be present in beef extract
- Use of a control tube (nirate free tube) will indicate if bubble is due to fermentation of suger--if bubble is bigger in nitrate broth--extra gas can be attributed to denitrification
