Microbiology Lecture Notes 7&8 – Flashcards

Yes! All organisms require elemental oxygen, 

• ELEMENTAL OXYGEN: An oxygen ATOM. The smallest individual part of the element oxygen - O. 
• This is the form that you'd find oxygen in when its incorporated into compounds, such as glucose - C₆H₁₂O₆
• This is different than MOLECULAR OXYGEN: An oxygen MOLECULE is the smallest part of the element oxygen that can exist in a free state - in this case a pair of atoms or O₂. This is the oxygen that is part of the atmosphere that WE breathe.
• This molecular oxygen is toxic to anerobic bacteria

Obligate aerobes

• Note: the bacteria stays at the surface of the media in order to obtain the oxygen it needs for survival. 

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• The electron transport chain takes place in the inner member of the mitochondria of the cell in the human body and in the plasma membrane in the bacteria. 

• The electron turns on the proton pump in the cell membrane of the bacteria to initiate or continue the electron transport chain, the goal of which is to generate ATP or energy for the cell. 

• The proton pump pumps protons in the form of 2 hydrogen ions.
• H+ H+

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Oxygen!

• This image does not show the process by which the singlet oxygen (_^1/2 O₂ here) is bound to the H₂₎ This is, however, the outcome in obligate aerobes such as ourselves. 

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• Singlet oxygen is the common name used for an electronically excited state of molecular oxygen (O2). 
• It results when an electron is deposited on a molecular oxygen during the process of the electron transport chain. 
• It is a FREE RADICAL

• It is a free radical, meaning that it is an unstable molecule that, in order to acheive stability, steals electrons from other molecules, making them unstable and creating a cascade of havoc. 
• It's debatable whether singlet oxygen and superoxide free radical are actually synonmous. I think they are for our purposes

• Superoxide Desmutase converts singlet oxygen to hydrogen peroxide by coupling it with water.
• O₂^*  + H₂0 = H₂O_{2  or hydrogen peroxide}

• Singlet oxygen is highly toxic. Hydrogen peroxide is toxic, but less so. 
• Singlet oxygen is coupled with water and the SOD enzyme to create hydrogen peroxide. 

• Which is a cytotoxin? 
• a. singlet oxygen
• b. hydrogen peroxide
• c. SOD
• d. both a and b

•  Which enzyme is oxogenic?
• a. SOD (from H₂O and O = H₂O_{2= hydrogen peroxide)}
• b. catalase (From hydrogen peroxide, it produces water and OXYGEN)
• oxygenic = oxygen producing
• c. peroxidase (from H₂O_{2 =} H₂O)

Oxygenic processes yield oxygen

Anoxygenic processes do not yield oxygen

• It is genetically determined. An organism that is an obligate or facultative anerobe will produce one or the other. 

• TWO. Facultative anerobes produce both SOD and catalase.
• Obligate anerboes produce neither. 
• Aerotolerant organisms produce SOD superoxide desmutase in response to singlet oxygen. 

• Diabetics tend to lack circulation in the extremities. Because blood flow is diminshed, oxygen delivery is dimished, making the environment hospitable to anerobes. Anerobes can begin killing the tissues, which increases further tissue death and gangrene. 
• Anerobes are one cause of gangrene.

• One should not stitch up a deep puncture wound such as this because the puncture may have introduced bacteria into the tissue. Anerobic bacteria will die if left exposed to atmospheric oxygen but will thrive if the would is closed.  

• By altering pressure, you increase the solubility of oxygen to force the blood to carry more oxygen and thereby deliver more oxygen into the tissue. More oxygen makes the body less hospital to anerobic bacteria.

• Pretty unlikely, because surface injuries will be exposed to oxygen making them inhospitable to anerobes. 

• What type of bacteria are responsible for most disease?
• a. Obligate anerobes
• b. Facultative Anerobes - This makes sense because these bacteria are very adaptable and because we are, to an extent, facultative anerobes. 
• c. Miroaerophiles
• d. Aerotolerant
• e. Obligate anerobes

Facultative Anerobes

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• 1. Obligate aerobe - need oxygen
• 2. Obligate anerobe - oxygen is toxic
• 3. Facultative anerobe - grow better with oxygen but can survive without
• 4. Microaerophiles - prefers small amount of oxygen, too much is toxic
• 5. Aerotolerant - no preference. 

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• Facultative anerobes grow better in the presence of oxygen but have strategies to survive without it. (E)

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Facultative anerobes (to an extent). 

• There is not enough oxygen to act as terminal electron acceptor.
• Our bodies demand for energy production exceeds our ability to take in oxygen to accept the last electron. 
• After glycolysis lactic acid is produced instead of the electrons entering the electron transport chain. 
• Lactic acid creates the burning sensation that arises with strenous excercise.  

Micro= small

Aero= air

Philes = loving

•  Microaerophiles like oxygen but can only tolerate it in a small concentration, less than normal atmospheric conditions. 

• None. The agar plate surface is in direct contact with atmospheric oxygen making it an inhospitable place for a microerophile to grow. Such an organism may well grow in a broth media. (B)

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A microaerophile

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Aerotolerant organisms (D)

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Aerotolerant.

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1. Carbon
2. Nitrogen
3. Sulfur 
4. Phosphorus
5. Trace Elements
6. Elemental Oxygen

• What type of bacteria is one likely to find growing in fridge?
• a. mesophiles- prefer temperatures close to the human body. 
• b. thermophiles - heat loving bacteria
• c. chemoheterotrophs - because they use organic materials as their carbon source (i.e. meat, bread, cheese, fruit) 
• d. autotrophs - inorganic molecules as carbon source

• An organism obtained from a can of green beans with a domed lid is most likely which of the following? 
• heterotroph - 
• Yes, it is consuming an organic source of carbon
• autotroph - 
• no, because green beans are an organic carbon source and autotrophs obtain their carbon from non-organic sources. 
• aerobic organism 
• no, because it is growing in a container in which it was not exposed to oxygen. 
• all of the above

• An AUTOTROPH - an organism that uses inorganic materials as a carbon source. 
• *Photoautotrophs use carbon dioxide as a source of carbon as well. They use light as a source of energy.

• Nitrogen is used in the production on non-essential (meaning we do not have to take them in because we make them) amino acids. 
• In the production of DNA, the nitrogenous base (ATCG) are nitrogen based. 

• While some bacteria obtain nitrogen through the consumption of organic protein, others obtain it from  atmospheric nitrogen.

• In addition to carbon and nitrogen, all living cells require:
• sulfur
• elemental oxygen
• phosphorus
• trace elements 

• Sulfur is most commonly used in the production of amino acids, some of which possess a sulfur side chain. 

• Phophorus or phosphate, is essential to the hydrophilic phosphate heads of the phospholidid bilayer of the cell membrane. 

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• Adenosine Tri Phosphate (ATP)
• Cell membrane, esp. the phospholipid bilayer would most quickly feel the effects of the absence of phosphate

• The majority of organism obtain phosphate from their environment either via organic or inorganic sources. 
• Note: Metachomatic Granules are storage inclusions of inorganic phosphate in a substance known as volutin. 

• Vitamins and Minerals that act as cofactors (minerals) and coenzymes (vitamins)
• Coenzymes are organic cofactors. 
• Minerals are, by definition, inorganic.  
• These trace elements are neeeded for their ability to activate or enable certain enzymes to function. 

• These mineral trace elements act as COFACTORS enabling enzymes to function.
• A cofactor is a non-protein chemical compound that is required for the protein's biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules" that assist in biochemical transformations. 

• Nothing. In and of themselves they do not do much for the bacteria. The prefix "Co" indicates that they work "with" something else. This "something else" is usually an enzyme. The cofactor (minerals) or coenzymes(vitamins) allows the enzyme to acheive functionality. 

• Cofactors allow an enzyme to be activated when and where it is needed.
• HCl does this for pepsinogen, making it pepsin, and functional.
• Hydrochloric acid is analogous to a cofactor a mineral trace element. 

• Folic acid is a B vitamin that complexes with the enzmye that facilitates the formation of the spinal cord in embyological development. 
• It is a coenzmye. Coenzymes are organic cofactors. 

• One can lengthen generation time for a given bacteria by manipulating the conditions of its physical and chemical enviroment, such as temperature, pH, oxygen availability.

The bacteria which causes tuberculosis

Mycobacterium tuberculosis, the exact time depends on the specific strain apparently. 

• 1. ASK: How many times did the organism double (N for number of times doubled) ?
• 4hrs x 60mins = 240 min/30min (GT) = 8 times
• Answer= the organism doubled 8 times. 

• 2. ASK: How many organisms will result if the original number of organisms is doubled N times
• 2>_1x4>_2x8>_3x16>_4x32>_5x64>6x128_7x>256_8x>512
• Answer =512
• This process can also be expressed and determined by the following equation: 
• F = I x 2ⁿ
• F= final number
• I= initial number of organisms (2)
• n= number of doublings. (8)
• ex. 512 = 2 x 2⁸
• F= 2 x (2x2x2x2x2x2x2x2)
• F= 2 x 256
• F= 512

• The number of organisms predicted to grow is the Generational Potenial
• This number assumes the organism will reach this potential which assumes that no bacteria die. 

• 1. Ask "How many doublings will occur?"
• 2 x 60 = 120/20 = 6 doublings will occur
• 2. Determine how many organisms will result in that amount of time using the following equation.
• F= I x 2ⁿ
• F= 3 x 2⁶
• F= 3 X (2x2x2x2x2x2)
• F= 3 x 64
• F = 192

• The first step is to determine how many doublings will occur in the given inubaction period. 
• Step 1. Convert the incubation period to minutes if the GT is in minutes
• e.g. 3 hours x 60 minutes = 180
• Step 2. Divide the number of minutes in the incubation time by the GT or doubling time
• 180/30 = 6 
• This is the number of doublings that will occur of the original organisms in 3 hours. 
• The next part is to determine how many organisms this will produce. 
• This is the equation used: F= I x 2 ⁿ
• N= number of doublings, 6 in this case. 

• The second step, after determination of N = number of doublings, is to determine how many organisms will result from said doublings. 
• To do this we can use the following equation. 
• F= I x 2ⁿ
• F is the final number of organism 
• I is the initial number of organism e.g.4
• N= is the number of doublings e.g. 6
• F = 4 x 2⁶
• F= 4 x (2x2x2x2x2x2)
• F = 4 x 64
• F= 256
• OR   
•  4 >_1x 8>_2x16>_3x32>_4x64>_5x128>_6x256 

• When an organism doubles under optimal conditions it may reach its  generational  potential.

• The equation assumes that an organism reaches its generational potential, or generates as many times as it possibly can in the given incubation period without a single bacteria dying. 

We graph bacterial growth logarithmically because it is difficult to graph population changes of such enormous magnitude using arithmetic numbers. 

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Bacterial growth is expressed as logarithmic growth.

• The logarithm of a number is the exponent to which another fixed value, the base, must be raised to produce that number. For example, the logarithm of 1000 to base 10 is 3, because 1000 is 10 to the power 3: 1000 = 10???10???10 = 103
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• Logarithmic tables make is easy to calculate how long one needs to incubate a given bacteria in order to obtain a certain number of organisms, for example, in vaccination manufacturing or antibiotic production. 

• Logarithmic tables would allow public health officials to determine how much of an organism causes symptoms and to thereby work backwards to determine the point of time origin of infection.
• To determine ID₅₀

A logarithmic scale

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1. Lag phase
2. Log phase
3. Stationary phase
4. Death phase/Decline Phase*

*Prof. McCleary uses "decline" the textbook uses "death". She also calls these stages, not phases.

• The microorganisms being born are equal to those dying. (also happens in stationary phase)
• There is no significant increase in the number of organisms. 
• Prepartory phase, production of enzmyes, acclimation to environment.

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A. Lag stage

B. Log stage (logarithmic stage)

C. Stationary stage

D. Decline stage

T stands for Time

L stands for Logarithmic number of organisms. 

• The number of organisms originally acquired. The closer to the ID₅₀, the shorter the lag phase.
• The generation time of the organism acquired. The shorter the generation time of the organism, the shorter the lag phase.
• The type of media will influence the duration of the  LAG phase.
•  e.g. If an organism is going from one host to another, the media is similar, so less acclimation time is needed. If an organism is going from a rich media to a sparser media, the LAG time will be longer.  

The preparatory phase of bacterial development is the LAG phase/stage. 

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• The LAG phase will be of a longer duration when one aquires only 5 organisms because those five organisms will have to double twice before they are able to produce symptoms in 50% of the population. 
• Note: Symptoms appear at the uncture at the end of the LAG phase/beginning of the LOG phase
• 15 organisms only have to double once to produce the same effect and because their first doubling will supersede the ID₅₀ this effect will produce symptoms in a greater proportion of the population. 

• The shorter the generation time of the organism, the shorter the lag phase will be. 

Symptoms first appear at the very beginning of the LOG phase. 

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• During the LOG phase antibiotics are most likely to be prescribed because symptoms become more defined and it becomes clearer that the patient is ill as a result of a specific organism. 

• The immune system kicks in towards the culmination of the log phase, but prior to its peak. It takes so long because must do all of the following before it can actively respond to the organism:
• see the organism's antigen
• make a memory of it (memory Tcells)
• multiply that memory
• make plasma cells that are capable of responding to it
• plasma cells must then make antibodies
• Antibodies then actively attack bacteria. 
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• No. Antibiotics kill some bacteria. Resistant organism are always left behind. The antibiotics take out a number of organisms and the immune system comes in and takes care of the rest. If it were not for the immune system these bacteria would replicate and cause illness. 
• The immune system cures. 

• You are ill with a bacterial infection. You take an antibiotic. A few weeks later you are better. Who should be credited with your wellness?
• a. antibiotic
• b. immune system

• During the lag phase and during the stationary phase, the number of organisms being "born" equals the number "dying"

• Virulance factors of organisms effect duration of stationary phase 
• e.g. Capsule -increases duration of stationary phase because it makes the organism more difficult to detect/attack
• Flagella increase duration of stationary phase because organism can leave inhospitable areas, therefore evading death
•  Non-compliance with antibiotic regime due to decrease/resolution of symptoms

• Noncompliance occurs most frequently in the STATIONARY PHASE because one is not getting worse. 

• Replase most often occurs during the stationary phase due to noncompliance with antibiotic regime. 

• A secondary infection is most likely to occur in the STATIONARY PHASE, because the immune system is already fully engaged in fighting the current infection

Decline.

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• During the initial section of rise of the log phase and the initial section of descent of the decline phase. This is because we do not feel "as sick" during these phases and so we tend to go out and be more sociable despite the fact that we are shedding organisms. 
• When we are most ill, we tend to stay in. 

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• The secretion of polysacchrarides.
• Biofilms are created by bacteria that have fimbriae. The organisms also create a polysaccharide secretions to which they can use the fimbrae (gram negative) to adhere. These secretions are similiar to those found in a slime layer, which is is also a sugar (polysaccharide) based substance.

Bacteria tend to form biofilms on indwelling medical devices

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• In a biofilm community the most fragile organism inhabit the inner area of the biofilm. They garner protection from this arrangment.

Biofilms are structured with holes between that allow an incoming flow of nutrients and an outgoing flow of waste products. 

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Quorum sensing. 

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• Quorum sensing protects bacteria. It's a strength in numbers strategy which allows bacteria to wait till they are of a certain population before they release their toxin or form their product, thereby not calling the attention of the immune system to themselves before they have been able to launch their "attack" 

• The more chemical an organism releases, the more attention he draws from the immune system.

Liquid agar!

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• 10ml was what we were told in class but that's actually the combination of the 9ml of liquid agar and 1ml of organism transferred.  (p. 171)

One must pour each dilution onto an empty pertri dish, incubate each for 18- 24 hours and then count the organisms that have grown.  

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Because there are too few to be statistically significant. (see final plate below)

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Number of colonies x  the denominator of the dilution factor.

• The first plate is TNTC
• The third plate in has 23 colonies, which is two few for statistical significance, as are the two that follow it.  
• The second plate from the left is the best for this task, which Prof. M. said should invovles btwn 70-200 colonies.
•  (Other sources say no lower than 30)
• The dilution for our optimal plate 100,000. Therefore the amount of bacteria per ml is the number of colonies seen in plate 2 x 100,000.
• If the original amount diluted we less than 1ml we would need to add zeros for each place value from one.
• NOTE: CFU in the image below means COLONY FORMING UNITS
• colony forming units are always expressed in parts per 1ml

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Advantages: Only counts living cells

Disadvantage: Time consuming, not only counting colonies but also waiting for them to grow

NOTE: Equation is given below

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• The final equation must be corrected for transfers smaller than 1ml. 
• TO DO THIS add as many zeros as the initial transferred amount is from the one's place.
• e.g. Given a 0.1ml transfer that yields a plate count of 54, at a dilution of 1/1000 parts
• Complete the normal equation 54x1000= 54,000 
• Then move the decimal one place to the right to correct for the 0.1ml (one decimal place to the left of 1ml) original dilution.
• 540,000 CFU (colony forming units) per mililiter.

Attend to:

• How much was transferred (1ml, 0.1ml, 0.01ml)
• What plate is being counted. 
• These will both effect the final equation tremendously. 

Filtration

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Filtration

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• No, filtration is only used to measure relatively small amounts of bacteria, such as would be found in drinking water source, not enormous amounts, like those that would be seen in sewer water. 

• Filtration. Filtration searches for coliform bacteria (coli as in colon, as in fecal contamination) in water sources. These are gram negative bacteria. 

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Placing the filter in a liquid media, an endobroth is common. 

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Filtration detects coliform bacteria, a gram negative bacteria. 

The common broth is called an endo-broth

• MPN means most probable number. 
• It is used primarily in food service industries. 

MPN is used in food service.

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0.01 ml

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• Microscopic counts use a grid, a slide, a stain and a microscope along with 0.01 ml of sample.

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A Microscopic count

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Turbidity

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• Light is passed through a sample, the amount of light that reaches the detector reflects how many bacteria are in the sample. The more bacteria, the less light hits the detector.
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Turbidity

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A spectrophotometer

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