Microbrial Growth Flashcards

Question

Where is Phosphorous found in cells and how do bacteria obtain it?

Answer 1

Phosphorous -found in DNA, RNA, ATP and membranes -DECOMPOSITION of organic sources or Po4^3- serves as sources of Phosphorous for bacteria

Answer 2

Sulfur -in Amino Acids (like Methionine and Cysteine), Thiamine and Biotin -Some bacteria use SO4^2- or H2S to obtain sulfur (combined sulfur and phosphorous represent 4% dry weight)

Answer 3

The Effect of O2 on Growth -Obligate Aerobes (require O2 for growth, and will only grow at TOP of tube; hence particles are only diffused at top) -Facultative anaerobes: prefer to use O2, but can also survive WITHOUT O2. hence more Growth will be at TOP of tube, and less growth at BOTTOM (hence high [ ] of particles at top and a few particles scattered to bottom. -Obligate Anaerobes - DO NOT USE O2; and microbes will grow only at the BOTTOM of tube -Aerotolerant anaerobes; They do NOT use O2, but grow EQUALLY throughout the tubes Microaerophiles: Require O2, but can only tolerate a Small amount of O2. (if there is too much or too little O2, it will NOT grow) . Hence most growth is MIDDLE of tube It is not That O2 is toxic, but rather the reactive species derived from O2. **Oxygen constitutes 20% of the dry weight of bacterium E.coli

Answer 4

O2: Reduction Products -O2 is capable of accepting 4 electrons, reducing it to water - 4 electron reduction steps for O2 progressively generate Superoxide, Hydrogen Peroxide, and the Hydroxyl radical plus water (throughout each strip you add 1 electron atom) (O2 + 1 e- --> O2-(superoxide). Then Superoxide O2- + 1e + 2H+ --> H2O2 (hydrogen peroxide). Then H2O2 + e- + H+ --> H2O + OH* (hydrogen radical) Then OH* + e- + H+ --> H2O)

Answer 5

Active respiratory chains produce ROS (reactive oxygen species) (seen in citric acid cycle, ETC) Electrons can Leak from the main path and directly REDUCE O2 to superoxide -ROS are also produced: 1) as necessary intermediates in a variety of enzymatic reactions 2) by ionizing radiation (ionize H2O to hydroxyl radicals + H's) quinones: transfer electrons from Complex 1 to III and can accidentally transfer e- to oxygen

Answer 6

Oxygen species that Harm organisms: 1) Singlet Oxygen 1 O2: an extremely reactive form of molecular O2 in which one of the electrons jumps to a higher orbital following energy absorption *** the Singlet Oxygen is an Excited state. NOT a free radical. Mostly a byproduct of Photosynthesis 2) Free radicals ** Superoxide; O2- O2- + O2- + 2H SOD--> H2O2 + O2 ** Peroxide anion: O2^2- 2 H2O2 Catalase---> 2 H2O + O2 (catalase test) H2O2 + 2H+ peroxidase-----> 2 H2O ** Hydroxyl Radical (OH*) -Formed by ionizing radiation and respiration (traces), very short half life (10^-9s) and MOST reactive. Can't be eliminated by an enzymatic

Answer 7

Toxic forms of oxygen need to be NEUTRALIZED by enzymes -Superoxidase dismutase (SOD) -Catalase -Peroxidase **Cells have one or the other (catalase or perodixase ) , but NEVER both Obligate anaerobe: SOD +Catalase Facultative anaerobe: SOD + catalase Obligate anaerobe; Neither (will not survive in presence of O2) **Aerotolerant anaerobe: SOD + peroxidase Microaerophiles: SOD +/- catalase (small amounts) (may or may not have catalse)

Answer 8

Potassium: 1% of dry weight Magnesium: 0.5 % of dry weight Calcium: 0.5% of dry weight All three serve as inorganic cellular cations and inorganic cofactors for certain enzymatic reactions

Answer 9

Trace elements: inorganic elements required in minute amounts that are generally used as Inorganic cofactors (ex: iron, copper, molybdenum, Zinc) -Commonly found in Tap water

Answer 10

Organic Growth factors: Essential organic compounds that an organism CANNOT synthesize and must be obtained from the enviroimet (ex: vitamins, amino acids, purines and pyrimidines) Organic growth factors are SPECIES-SPECIFIC because many bacteria can synthesize compounds and do NOT require outside sources

Answer 11

Vitamins, Amino Acids are organic growth factors for humans. (humans can make pyrimidines and purines)

Answer 12

because if you are not at OCEAN depths; you will NOT reach temps above 120 degrees Celsius

Answer 13

REVIEW (PLAY RECORDING FOR ANSWER

Answer 14

Cysteine, Methionine, Biotin and Thiamine are molecules where the sulfur would be in. 32P?

Answer 15

- Microorganism typically live in communities called BIOFILMS in which cells (either single or diverse species) are embedded within an EPS (Extracellular Polymeric substance) (informally called Slime) -3D structure of biofilms was NOT well appreciated until the development of confocal microscopy -Biolfilms are NOT a thick uniform monolayer (they grow in 3d structure that allow water to pass through thin layer) Quorum sensing: the ability of bacteria to coordinate gene expression with other bacteria via signaling molecules. they respond to local production density Have two distinct programs (switch between high-density group growth and low-density individual growth) (biofilm development and dispersion)

Answer 16

Biofilm features: -Usually attached to a surface -May facilitate transfer of genetic information -Work cooperatively -Sheltered from harmful factors (desiccation, antibiotics, immune system) -10-1000 less susceptible to antimicrobials

Answer 17

because cells in the center grow slower and are processed by the negative charge of the bio matrix (hence cells that grow slower are more resistant to antibiotics)

Answer 18

The Biofilm Life Cycle: 1) Attachment 2) Growth 3) Dispersal Process: 1) Free-floating (planktonic) bacteria encounter a surface, attach and produce extracellular polymeric substances (EPS) 2) EPS production allows the emerging biofilm community to develop a complex, three-dimensional structure 3) Biofilms propagate by detachment of small or large clumps of cells, or by a type of "seeding dispersal" that releases individual cells.

Answer 19

Biofilms can form on medical implants and Cause INFECTIONS Strategies to prevent biofilm formation 1) incorporate antimicrobials into surfaces that biofilms might form (by impregnating sliver) 2) Block quorum sensing (ongoing research) (involves preventing organism from turning on gene to form biofilm) 3) Lactoferrin used (binds/depletes iron) to STIMULATE surface motility and prevent biofilm formation

Answer 20

Culture Medium: Nutrient material prepared for microbial growth in the laboratory Requirements; -Must initially be sterile -contain appropriate nutrients, sufficient moisture, correct pH and osmolarity -Must be incubated at appropriate temperature and O2 concentration Culture: 2 meanings 1) Verb: the act of multiplying microbial organisms by letting them reproduce in predetermined growth media under controlled laboratory conditions 2) Noun- Microbes that grow and multiply in or on a culture medium Ex: bacillus anthracis

Answer 21

Liquid Media: Broth Solid Media: Same as broth but contains a solidifying agent The most common solidifier is AGAR (complex polysaccharide extracted from marine algae/seaweed species) -Completely solid medium (1.5-2% agar) -soft agar (less than 1% agar)

Answer 22

Important properties of Agar: - **it MELTS between 80-90 degrees Celsius - Once melted, it DOES NOT Solidify until it reaches 40 degrees C -Cannot be catabolized (broken down) by the vast majority of bacteria -Originally used as a Food thickener (Angelina Hesse, 1880s) No satisfactory substitute has been found Gelatin not considered food thickener because organisms can use it as energy (carbon) source and it liquifies in 37 degrees C (most pathogens grow at 37 degrees C) (more pure algar are closer to 90 degrees C) REVIEW

Answer 23

Although agar growth media is usually poured into Petri dishes, it can also be put into slants and deeps -Slant: used to propagate bacteria Broth: Deep: REVIEW

Answer 24

Agar slant: 1) pour tube at angle 2) Streak agar with inoculated loop and incubate Agar Deep: -Stab inoculum into solid medium and then stab culture

Answer 25

Soft agar deeps are used for MOTILITY tests -soft agar (less than 1% agar ) contains Tetrazolium dye (used to visualize cells) (reduced in living cells) Very motile: if the entire tube is turbid (indicates bacteria has moved away from stab mark and is Very motile. non motile; if the stab mark is visibly clear and (tube not turbid), bacteria are nonmotile motile ; if there are still a little visible parts of tube, and some turbidity; bacteria are motile REVIEW

Answer 26

Chemically Defined Media: Nutrient material whose EXACT chemical composition is known Must contain an ENERGY SOURCE* along with C^, N?, S, P, (K, Mg, CA) organic growth factors and trace elements that the microbe requires -Unless phototrophic* (don't need carbon as energy source; uses light) Unless Autotrophic (no need for carbon)

Answer 27

A chemically defined medium for growing a Chemoheterotroph like Escherichia coli; Glucose adds 5.0 grams Ammonium phosphate, monobasic adds 1.0 grams (NH4H2PO4) Sodium Chloride (NaCl) adds 5.0 grams Magnesium Sulfate (MgSO4 . 7H2O) adds 0.2 grams Potassium phosphate, dibasic (K2HPO4) adds 1.0 grams Water adds 1 liter

Answer 28

Fastidious organism: one that has complex nutritional requirements (like many growth factors) ex: chemohetrophic bacterium like Neisseria requires carbon and energy sources, Salts, amino acids, organic growth factors and Reducing agent, and water

Answer 29

Complex Media: nutrient material whose Exact chemical composition is NOT known -Routinely used for heterotrophic bacteria and fungi -Made from protein hydrolysates or meat/yeast extracts -Protein fragments (PEPTONES) provide energy, carbon, nitrogen, and sulfur requirements -Meat and Yeast extracts provide vitamins and other organic growth factors (also supplement organic and nitrogen sources) Composition may vary slightly from batch to batch ***Many different formulations: LB, YEPD, 2xYT, Nutrient, SOC

Answer 30

Composition of Nutrient Agar (complex media for growth of Heterotrophic Bacteria); - Peptone (partially digested protein) adds 5.0 grams to media -Beef extract adds 3.0 grams to media -Sodium chloride adds 8 grams to media -Agar adds 15 grams to media -Water adds 1 liter to media

Answer 31

Anaerobic Culture Media: **Oxygen must be REMOVED from the Media Can be done by: 1) Heating: -Liquid media can be heated/autoclaved to drive off dissolved O2, then tightly sealed. 2) Reducing media -contains a chemical, such as sodium thioglycolate [HS-CH2COONa], cysteine or glutathione that removes O2 by reducing it to water Other methods used to remove O2: Bubble Nitrogen to liquid media, so liquid can replace Oxygen

Answer 32

Methods to incubate plates oxygen free: 1) Anaerobic Jar 2) Oxyplates 3) Anaerobic Chamber

Answer 33

Anaerobic Jar: -Useful if brief exposure to oxygen is NOT lethal process; inoculated plates are placed in jar and water is added to gas generator envelope before jar is sealed to create anaerobic environment REVIEW

Answer 34

Oxypaltes -medium contains an enzyme (oxidase) that uses an organic substrate (Lactic acid) to REDUCE Oxygen to WATER -can be opened and RESEALED 4-5 times and will still regenerate anaerobic conditions -useful if BRIEF exposure to oxygen is NOT lethal

Answer 35

Anaerobic chamber: 90% N2 5% H2 +/- 5% CO2 process: a hydrogen gas mixture is circulated through a heated pallidium catalyst to remove O2 by formic water (H2O) REVIEWWh

Answer 36

Special Culture Techniques; for bacteria with UNUSUAL growth requirements: 1) Bacteria that Do NOT grow on artificial media -Myobacterium leprae (leprosy); propagated in armadillos (natural reservoir) -Treponema pallidium (syphilis): Pathogenic strains grown in rabbit testicles. some nonpathogenic strains can be grown on synthetic media -Other obligate intracellular bacteria (rickettsias, chlamydias, etc) won't grow on artificial media 2) Bacteria that Thrive at HIGH CO2 levels: Capnophiles: Grow best at high CO2 concentrations. Similar to environment of the intestinal tract, respiratory tract, and other tissues

Answer 37

Equipment for producing CO2 Rich environments 1) Candle Jar (contains lid, glass jar, tubes with liquid media, candle, and petri plates with solid media (inverted) 2) CO2-generating packer: contains petri plate with bacterial culture, gas generator. You use CO2 packet, seal plates in packet and release chemical that produces CO2. -Atmosphere is 20% O2 and .03% CO2 -Candle stops burning at 17% O2 and 3% CO2 In most labs, sealed incubators are connected to a CO2 tank and levels are controlled electronically

Answer 38

Biosafety levels Ranked from one to four and are selected based on the agents/organisms on which the research or work is being conducted. Each level up builds on the previous level, adding constrains and barriers Levels: 1. Well-characterized agents or minimal potential hazard to lab personnel and environment--Labs are not isolated from general building, work performed on open lab tops, personal protective equipment (PPE) such as: eye protection, gloves, and a lab coat worn as needed 2. Agents of *moderate* potential hazard (influenza A, Salmonella etc)- Equipped with a class II biosafety cabinet when procedures may lead to production of infectious aerosols. *hand wash, eyewash station and self-closing doors* 3. Indigenous or exotic agents which may cause serious or potentially Lethal disease after inhalation (ex: Mycobacterium tuberculosis)- ALL work performed in biosafety cabinets to prevent airborne tramission. Lab should be Negatively pressurized and equipped with filters to prevent pathogen release. Enter lab through 2 self-closing doors 4. Dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections, agents which cause severe to Fatal disease in humans for which vaccines or other treatments are NOT available (ex; Ebola)-SEALED lab, **intake and exhaust air is HEPA filtered twice, personnel wear space suits** (only a handful in the US)

Answer 39

Biosafety Level 4 (BSL-4) Labs: 1. CDC (center for Disease Control and Prevention 2. NIH (National institute of Health) 3. Defense Military Lab 4. George State University

Answer 40

Selective Media: Suppress the growth of Unwanted bacteria and encourage the growth of desired microbes -Sabouraud's Dextrose Agar (sugar); pH of 5.6 Discourages bacterial growth. Used to isolate fungi. Made more selective by addition of Chloramphenicol (antibiotic used to kill bacteria) It was created by and is named after Raymond Sabouraud in 1892 (composed of 40 g/L dextrose, 10g/L peptone, 20 g/L agar at pH 5.6)

Answer 41

Differntial Media: Used to Distinguish colonies of a desired organism Blood Agar: used to distinguish bacteria that destroy red blood cells (HEMOLYSIS) Hemolysis appears as an area clearing around a colony . Streptococcus progenies (5 % sheep blood; organisms lyse blood and produce zone of clearing around colony) **also useful for cultivating fastidious organisms (since blood has a lot of organic factors, cells can use)

Answer 42

Media Both Selective and Differential: -Distinguishes colonies of a desired organism and INHIBITS the growth of other microbes -Mannitol Salt Agar: Used to select for and distinguish Staphylococcus aureus -pH indicator changes color when mannitol is fermented to acid.

Answer 43

MacConkey Agar: Used to distinguish and select for GRAM NEGATIVE cells that Ferment Lactose -Bile salts and crystal violet discourages growth of gram-positive bacteria -Lactose plus pH indicator: Lactose fermrenters produce pink or red colonies, Nonfermenters are colorless (NEUTRAL RED is colorless ant any pH greater than 6.8) Enterics: family of organisms that are rod-shaped when produce acid (found in intestine of animals) Enterics that can ferment are called COLIFORMS and are usually considered nonpathogenic in intestinal tract (ex: Escherichia coli) The enteric pathogens, Salmonella and Shigella are Unable to ferment lactose.

Answer 44

Bismuth Sulfite Agar -Highly SELECTIVE medium used to isolate Salmonella species, particularly** Salmonella typhi**from feces Bismuth sulfite Bi2 (SO3)3 INHBITS most gram-postiive bacteria and most gram-negative bacteria EXCEPT Salmonella -Agar also contains Ferrous Sulfate. S. typhi is the ONLY Salmonella that reduces it to Hydrogen Sulfide Differential on the basis of Hydrogen sulfide production -

Answer 45

Enrichment Culture: Used for preliminary isolation that favors the growth of a particular organism -Similar to selective medium, But designed to increase very small levels to detectable levels -Usually performed in Liquid -Does NOT contain any inhibitors to inhibit the growth of other organisms -Used mainly for Fecal and Soil samples -Enrichment increases the likelihood of positive identification Easy Clostridium enrichment : take solid sample and boil it. Endospores go through and take endospore and incubate it. Select for clositridum - Phenol enrichment: REVIEW

Answer 46

Pure Culture: contains a SINGLE microbial species -most clinical and environmental specimens contain several different microorganisms -To obtain a pure culture, individual organisms must be ISOLATED -The most common method of isolation is the STREAK PLATE, in which a sterile inoculating loop is inserted into a sample and streaked onto a plate in a pattern, to obtain individual colonies

Answer 47

NO, because you would only be able to isolate a few from a billion (might want to use enrichment method instead) REVIEW

Answer 48

Preserving Bacterial Cultures: Refrigeration can be used for Short-term storage (days-weeks) -2 methods for Long-term storage (years) -Deep freezing: (80 degrees C) -Lyophilization (freeze-drying); Frozen (-54 to -72 degrees C) then sublimated (solid to gas) in a vacuum. When lyophilization is complete, the impulse neck is sealed under the vacuum

Answer 49

Bacterial Division -Most bacteria divide by Binary fission Alternative means: 1) Budding (observed in some members of the Planctomycetes, Cyanobacteria, Firmicutes) (cell frm small bud, will enlarge and pinch off parental cell) 2) Conidiospores (some filamentous bacteria), reproductive asexual spores carried at the tips of filaments (ex: penicillium have conidiospores -Fragmentation: (few filamentous bacteria) filaments break off and initiate growth of cells.

Answer 50

Binary fission 1) cell elongates and DNA is replicated 2) Cell wall and plasma membrane begin to constrict 3) Cross-wall forms, completely separating the Two DNA copies 4) Cells separate (components; cell wall, plasma membrane, DNA (Nucleoid) ex: Bacillus licheniformis undergoes binary fission

Answer 51

Generation/Doubling Time: time required for cell to divide/for population to double -Generation time varies considerably: -E.coli divides every 20 minutes (quick) - Most bacteria divide every 1 to 3 hours -Some bacteria require over 24 hours to divide

Answer 52

E.coli, 20 generations (7 hours), 1 cell becomes 1 million cells

Answer 53

Logarithmic Representation of Bacterial Growth: The number of cells in a bacterial generation can be expressed as 2^n (only applicable if you have 2 cells), where n is the number of doublings that have occurred. ex; if have 1 cell, double it will have 2 cells (1 generation) if double 2 cells have 4 cells (2 generations); if you double 4 cells you will have 8 cells (3 generations) and so on.. (2^1= 2 cells, n= 1 doubling, hence double 1--> 2)

Answer 54

Bacterial Growth Curve (plotted logarithmically and arithmetically) arithmetic curve: NOT a good way to represent cell growth because it is hard tossed small changes in number of cells. Logarithmic curve: it is better to convert to log numbers to see changes in the number of cells. Take log of numbers form straight. line in graph

Answer 55

Bacterial Growth Curve: Phases of Growth (cells inoculated into liquid culture) Lag phase; cell number does Not increase Log phase: cells start to double; number of live cells are greater than dead cells Stationary phase: cells alive= cell death -runt out of O2, and nutrients Death phase: number of cells divide are less than cells that die Slopes and growth phase are media and bacterial species dependent

Answer 56

Four phases of Bacterial Growth; 1. Lag Phase: -period of adjustment to new conditions -little or NO cell division occurs, population size does NOT increase -intense phase of metabolic activity, in which additional organisms grow in size -May last from one hour to several days. 2) Log Phase: -cells start to divide and generation time is constant -Period of MOST RAPID growth ***Number of cells produced > Number of cells dying -cells are at the HIGHEST metabolic activity -Cells are MOST SUSCEPTIBLE to adverse environmental factors at this stage -Radiation -Antibiotics 3) Stationary Phase: Population size stabilizes **Number of Cells produced balances dying cells (equal)** Factors that slow down microbial growth: -Accumulation of byproducts (ex: Acids) -Insufficient nutrients -Insufficient Oxygen 4) Death or Decline Phase: -Population size begins to decrease - **Number of cells dying > Numbers of cells produced -cell number decreases at a logarithmic rate -a tiny fraction of cells may remain alive for a long period of time.

Answer 57

Measuring Growth: Direct methods- count individual cells -Plate counts -Filtration -MPN -Direct microscopic count Indirect methods- measure the effects of bacterial growth Turbidity Metabolic activity Dry weight

Answer 58

Plate Count: Frequently used method of measuring bacterial populations. Spread plate with a sample and count number of colonies Assumptions: -Each colony originates from single bacterial cells (NOT always true because some bacteria grow on clumps, filaments -Original inocules is Homogenous (suspension is equally mixed) Advantages: Measures VIABLE cells Disadvantages: takes 24 hours or more for visible colonies to appear (Long time) -only counts between 25 and 250 colonies are Accurate -Must perform SERIAL DILUTIONS (the concentration of cells cannot be too high or you will not get individual colonies) Plate Count (Direct measurement of bacterial growth )

Answer 59

Spread Plate method: 1) Sample (0.1mL) is Poured onto solid medium 2) SPREAD sample evenly over the surface (amount of liquid on plate is limited) 3) Plate will be INCUBATED until bacterial colonies grow on the surface of the medium.

Answer 60

Each subsequent plate will have 1/10 cells for dilutions Calculation: Number of colonies on plates reciprocal dilution of sample = number of bacteria/ml Ex: If 54 colonies are on a plate of 1:1000 dilution, then the count is 54x 1000= 54,0000 bacteria/ml in sample REVIEW

Answer 61

SERIAL DILUTIONS are used with plate count method to measure numbers of bacteria Serial dilution of cultures: A series of 1:10 dilution is made (along each step increases by factor 1/10) However, different dilutions can be employed (not all 1/10s) To calculate number of viable bacteria in original sample: Number of colonies/Volume plated x 1/dilution

Answer 62

A POUR PLATE is a second type of plate count The Pour plate method 1) inoculate EMPTY plate 2) Add melted nutrient agar 3) Swirl to mix 4) Colonies grow on AND In the solidified medium Meanwhile steps of Spread plate method 1) inoculate plate containing SOLID medium, 2) Spread inoculum over surface evenly, 3) colonies grow ONLY on Surface medium also use 0.1 ml of bacterial dilution) -Pour plate method can use 1.0 or 0.1 ml for bacterial dilution

Answer 63

Pour plate method Disadvantages -Heat sensitive microbes may be harmed -Embedded colonies are NOT easily transferable -some differential plates require surface colonies for diagnostic morphology -Some obligate aerobes will grow poorly (and thus be much smaller) if deeply embedded. Advantages -Can be used on aerobes and anaerobes -embedded- Bacteria cannot move, can add more culture to plate REVIEW (more sensitivity, more accurate ?) REVIEW

Answer 64

Counting by Filtration (Direct method) -Used to measure DILUTE bacterial samples -Example: Fecal bacteria in a lake or in ocean water. A 100 ml or more sample is filtered to retain bacteria -Filter is then transferred onto a Petri dish to incubate and count colonies -Filter is saturated with liquid medium to enumerate coliform (count 124 bacteria per 100 ml)

Answer 65

Most Probable Method (Direct method) -Used mainly to measure bacteria that will NOT grow on solid medium Process: -Dilute a sample repeatedly and inoculate several broth tubes for each dilution point -Count the number of positive tubes in each set (in experiment with 3 sets, and 5 tubes for each set; set 1 used 10 ml inoculum and had 5 positive tubes; set 2 used 1 ml of inoculum and had 3 positive tubes; set 3 used 0.1 ml inoculum and had 1 positive tube) -You then compare results of MPN count method for experiment with a statistical table (have combination of positives, MPN index/100ml , and 95% confidence limits (upper/lower) in table Statistical method: Determines 95% probability that a bacterial population falls within a certain range

Answer 66

Direct Microscopic Method Count: -A bacterial suspension (0.01 ml) is placed on a microscope slide with a special grid (Petfroff-Hausser cell counter) -Stain may be added to visualize bacteria -cells are counted and multiplied by a volume factor to obtain concentration Advantages: NO Incubation time required Disadvantages: Cannot always distinguish between live and dead bacteria -Motile bacteria are difficult to count -Requires a high concentration of bacteria (10 million/ml)

Answer 67

Petroff-Hausser Chamber slide: composed of Grid with 25 large squares -cover glass -slide Process: 1) Bacterial suspension is added on grid and fills the shallow volume over the squares by Capillary action 2) cross section of a cell counter 3) microscopic count 4) The volume of fluid over the larger square is 1/1,250,000 of a milliliter

Answer 68

Direct Microscopic count: Number of bacteria/ml = number of cells counted/volume of area counted ex: have 14 colonies, calculation; 14/8x10^-7 = 17,500,000 bacteria/ml

Answer 69

Direct methods : 1. Plate count 2. Filtration 3. MPN (most probable number) 4 Direct microscopic Count

Answer 70

Indirect methods: 1) Turbidity 2) Metabolic activity 3) Dry Weight

Answer 71

Turbidity (describes cloudiness of fluid; Indirect measurement) -Media becomes turbid as bacteria multiply -Determine % transmission or absorbance with Spectrometer **How are these values correlated to cells/ml? -You take the number from culture, and take another sample from culture, do plate count? Advantages: NO incubation time required Disadvantages: Cannot distinguish between live and dead bacteria -requires a High concentration of bacteria (10 to 100 million cells/ml)

Answer 72

Turbidity -Absorbance is a Logarithmic expression of percent transmission -Absorbance/optical density= 2- log % Transmittance Log 100 = 2 Log 1= 0 The absorbance is used to plot bacterial growth on graph and when plotted vs time, will form an approximately Straight line Linear range of absorbance: 0.1 to 1 (you can get absorbance of higher than 2 because of transmission that is less than 1)

Answer 73

Metabolic Activity: As bacteria multiply in media, they produce certain metabolic products that can be measured: -Carbon dioxide -Acids -method assumes that amount of metabolic product is in Direct proportion to cell Number (more metabolic product seen, more number of cells) Apparatus for Measuring production of CO2 during fermentation (Stained water drop, water brewer's yeast carbohydrates)

Answer 74

Dry Weight (Indirect method) -Best for measuring FILAMENTOUS bacteria or fungi Process: Cells in liquid media are centrifuged. Resulting cell pellet is desiccated (dry pellet) and weighed Wet weight and volume of cells after centrifugation measurements can also be made. -DOES NOT distinguish Live and Dead cells. (advantage; quick process)