micro sheet # 5

** Growth & Culture of Bacteria
* Optical density: it's the reflection of light from suspension of organism, this is reflection of the count of bacteria.
* Count of bacteria in billion so we use it in log just to make the curve possible and decrease the number.
The growth curve of bacteria


The growth curve of bacteria can be divided to 4 major phases: lag phase, log/exponential phase, maximum stationary phase, decline/death phase.
But there are actually 2 minor phases:
1_ if you look at the first arrow you notice it’s not a straight line, it start to curve >>> this is called acceleration phase, organism start to grow at an accelerated rate, it start modifying but it doesn't reach log/exponential phase yet.
2_look at the second arrow, we see there is a sort of delay >>>this is called retardation phase.

So 4 major phase and 2 minor, but al are use to study the kinetic of bacteria

The 4 major phase:
1_lag phase:
-It is the period where the individual bacteria are maturating and not yet to divide and multiply, it synthesis enzymes, RNA, proteins. So in this phase the bacteria are not dormant
-we count only the doublings (new generation).
-the count doesn't increase.

2_log/exponential phase:
-is a period characterized by cell doubling regularly and growing by genetic condition.
-rate of exponential is expressed as >>> generation/doubling time: is the time needed for the number of bacteria to be doubled.
And that’s the way to study bacteria kinetics.

3_stationary phase:
-the growth rate slows as a result of depletion of nutrient and accumulation of toxic product >>> inhibition of metabolic activity.
-lack of biological space.
-the phase is a constant value as the rate of bacterial growth is equal to the rate of bacterial death.

4_death phase:
The number of viable cells decreases geometrically (exponentially), essentially the reverse of growth during the log phase.


** Techniques for isolating Bacteria
1_Pure culture:
-we have many organisms grow in one media, but we should pick each organism alone and transfer it to a new media to initiate a pure culture.
-so simply picking out a single individual and put it in a special media to initiate a culture.

2_Streak plate:
-Here we use a tool called loop. It is made in the form of circle with a varies volume to carry certain volume of bacteria.
-it can be wire or plastic, the plastic one is disposable but the wire can be use it more than one after sterilize it by direct flame until it has a red glow.
-streak the organism by the loop an incubation for a while and the bacteria will grow.

3_Pour plate:
-just pour the content that contain the organism in the plate and that’s why we call it pour plate.

4_Membrane filter:
-the filter has a specific size of pores which retain the organisms and permit the fluid to pass through.
-filter which use for bacteria >>> 0.45 pores micron ,remember that most bacteria rang in size from 0.5 to 2.0 micron, so this filter will not allow the passage of bacteria.
-the water is filtered through a membrane filter and the filter pad is then transfer red to a plate of bacteriological medium. Bacteria trapped in the filter will form colonies.
** We can measure growth indirectly by measuring the cell mass of bacteria and this achieved by :
1_Direct physical measurement of dry weight, wet weight or volume of cells after centrifugation. Measurement of total cell yield in cultures.

2_Direct chemical measurement of some chemical composition like nitrogen, total protein, total DNA content. Measurement of total cell yield from very dense cultures for nitrogen and protein.

3_Indirect measurement of chemical activity such as the consumption of O2 or CO2 or any label substances. Requires a fixed standard to relate chemical activity to cell mass.


4_Turbidity measurement:
-The most accurate of all these measurement.
-we use standard tubes containing a known numbers of organisms and determine there optical density, and we take unknowns and determine there optical density and then make comparison between them.
-so turbidity whish use spectrophotometer can be utilized to determine the mass of bacteria.

** Methods for measurement of cell number
Note: medical microbiology is interested in cell number.
The industrial food is interested in cell mass.

— Measuring techniques involve direct counts, visually or instrumentally, and indirect viable cell counts.
— 1_Direct microscope counts are possible using special slides known as counting chambers. Dead cells cannot be distinguished from living ones. Only dense suspensions can be counted (>107 cells per ml), but samples can be concentrated by centrifugation or filtration to increase sensitivity..
2_Electronic counting chambers count numbers and measure size distribution of cells. For cells the size of bacteria the suspending medium must be very clean. Such electronic devices are more often used to count eukaryotic cells such as blood cells not for bacteria.
3_Indirect viable cell counts is considers as the best method
-If we have a preparation of organism, we can prepare a serial dilution of that organism.
- We prepare a number of tubes with the same amount of diluent.
Ex.:-
- We add 0.5 ml of diluent in all tubes and 0.5 ml of the organism in the first tube. And we will obtain the ratio of 1:2.
- We pour 0.5 ml from the first tube into the second one, thus having a ratio of 1:4 then the third one with a ratio of 1:8 ….etc
- This is two-fold serial dilution.
- We cultivate all of the tubes; the growth of bacteria from the first tube would be so heavy that we wouldn’t be able to count it. The growth from the second tube is less thick than the first one but still difficult for counting. As we move forward, it would be easier for us to count the bacteria.
- Let’s assume that we decide to count from the 1:8 ratio, each colony (which is a collection of organisms growing in agar and it contains about one thousand organisms) usually originates from one colony-forming unit.
-we take the last tube and culture 3 plates; we count the colonies in each one and take average …. NUMBER of organisms = average X dilution factor.

-Advantages of the technique are its sensitivity (theoretically, a single cell can be detected), and it allows for inspection and positive identification of the organism counted.
-Disadvantages are only living cells develop colonies that are counted.
** Generation Time and Growth Rate:
In the laboratory, under favorable conditions, a growing bacterial population doubles at regular intervals. Growth is by geometric progression: 1, 2, 4, 8, etc. or 20, 21, 22, 23.........2n (where n = the number of generations).
When growing exponentially by binary fission, the increase in a bacterial population is by geometric progression. If we start with one cell, when it divides, there are 2 cells in the first generation, 4 cells in the second generation, 8 cells in the third generation, and so on. The generation time is the time interval required for the cells (or population) to divide.
G (generation time) = (time, in minutes or hours)/n (number of generations)
G = t/n
t = time interval in hours or minutes
N0 = number of bacteria at the beginning of a time interval
N = number of bacteria at the end of the time interval
n = number of generations (number of times the cell population doubles during the time interval)
N = N0 x 2n (This equation is an expression of growth by binary fission)
Solve for n:
Log N = log N0 + n log2
n = log N – log N0
log2
n = log N0 – log N
.301
n = 3.3 log N0/N
G = t/n
Solve for G
G = t
3.3 log b/B
Example: What is the generation time of a bacterial population that increases from 10,000 cells to 10,000,000 cells in four hours of growth?


G = t_____
3.3 log b/B
G = 240 minutes
3.3 log 107/104
G = 240 minutes
3.3 x 3
G = 24 minutes
*** Bacterial genetics
-It is so important because most of our knowledge regarding molecular processes that take place, inheritance, gene expression here the result of studies done in bacteria.
-it has an important application in human science in general and in medicine in particular
- The rapid development of molecular genetics, in conjunction with the ability of scientists to manipulate and transfer genes, has elicited considerable interest among physicians, scientists, and the public in general.
-The ability to manipulate the hereditary material of microorganisms and even our own species has far reaching consequences.

- In biological system there are 2 sources of variation :
1- Mutations, things differ because of mutations.
2- Transfer of genetic material (DNA).

**Mutation:

-Mutation is defined as any change in the nucleotide sequence of a gene.
- gene is the unit of heredity, the gene is composed of nucleotides , each three nucleotides form codon which cods one amino acid and the amino acid can be coded by more than one codon ; that's why there's variations in heredity .
-change in the nucleotide sequence may result from base substitutions, base deletion, base insertion, base rearrangement.

-2 types of change in nucleotide:
1_transition: is a point mutation that changes a purine nucleotide to another purine (A ↔ G) or a pyrimidine nucleotide to another pyrimidine (C ↔ T).
2_transversion: refers to the substitution of a purine for a pyrimidine or vice versa.

- Under natural conditions DNA is subjected to a mechanisms that corrects these changes during replication of DNA, if an error took a place, (SOS) enzymes ( repair enzymes ) will try to repair this error and act to maintain cell sequence ,that’s why mutations in DNA are not common , because of the presence of the proofreading mechanism .

- After copying the strand of DNA an enzyme goes over the new strand and read it , if it contains errors usually these errors will be corrected this is called the SOS or" proofreading mechanism" which operates in DNA ,but this mechanism doesn't operate in RNA that’s why errors are more common among RNA viruses in compared with DNA viruses .
- In eukaryotes it's DNA that represent the genetic code but in viruses it's RNA.
-The consequence frequency of mutation in DNA is less than in RNA, its in the range of 10^-8 – 10^-11 , approximately in the average of 10^-9 .i.e one error or one change in nucleotide takes place in the replication of every 1000 million nucleotides .
- Because we have large number of cells, and large number of bacterial pollution so this number can be generated in a short period of time.
-these mutations accumulate in bacteria; the significance is that these mutations can be passed from one generation to the other.
- So if this mutation codes for a resistant to an anti-biotic or production of a toxin this can be acquired by any generation, that’s why mutations among bacteria are important because they can pass from one generation to the other.

**test of mutations depending on their functions:-
1- Silent mutation: when the nucleotide change leads to the same codon that cods the same amino acid despite the change of the nucleotide sequence (both sequences cods for the same amino acid).

2- missense mutation: leads to a change in the amino acid (change in he structure) with or without a change in the function .so an amino cid in the protein that result as a consequence mutation may perform the same function of the original amino acid or it mayn't, but this is a real mutation with or without a change in the function.

3- Nonsense mutation: is the mutation that leads to non-coding codon, no amino acid is coded by this codon , which may result in termination of synthesizing of protein because this leads to a non coding gene .

4- frame-shift mutation: the mutation that shifts the frame of the coding sequence by one or two or three amino acids . if one or two amino acids changes, the whole codon sequence will change so the frame will be shifted for the whole gene , but if the mutation involves three or multiples of three nucleotides then the frame won't shift but it result in addition of a new amino acid in the structure. But the codon on the coding sequence remains the same because each three nucleotides codes for an amino acid. If we delete three amino acids ,or if we add three amino acids or multiples of three the frame will not shift despite it’s a real mutation but the frame will stay the same , the frame shift if the change involves one or two amino acids or multiples of two but not three or multiples of three .

5- Null mutation: is the mutation that nullifies the gene (the whole gene is destroyed) and this usually takes place in gross agents so a change in the nucleotide's sequence that result in the loss of the function of the gene as a whole . So nullification of the whole gene takes place.

6- Suppressor mutation (intragenic or extragenic): involves the same gene, the mutation that takes place in a sequence which leads to the suppression of a previous mutation, if its an insertion- for example deletion will result in a suppression of the previous mutation and Vic versa and it affects the same gene (The effect of the previous mutation is cancelled as a consequence of the new mutation).

- Mutation can be spontaneous or induced in vitro by certain physical or chemical agents and as we mentioned before that mutations can vary anywhere from 10^-4 – 10^-11. But generally about 10^-7 .its rare to have such frequent mutations, 10^-4 is very common in molecular biology, and this takes place mainly in viruses, it undergoes such frequent mutations, but when we have hot spots (genes that are known to undergo frequent mutations) the mutation can be in frequency of 10^-4 , but usually its in the range of 10^-11 so DNA mutation is relatively rare .

-What causes spontaneous mutation?
-The cause is a process that promotes erroneous hydrogen bonding between the nucleotides .under natural conditions adenine makes hydrogen bonds with thiamine and guanine makes h- bonds with cytosine.
-A phenomenon known as "tautomerization" which is moving around of electrons in the nucleotide's sequence can enhances erroneous h- bonding. Instead of making h-bonds with adenine, thiamine bonds to cytosine -for example- which is a wrong type of h- bonding.
That is known to a physical state of nucleotide, there are two physical states of nucleotides, the first is known as keto form, where h- bonding is proper like thiamine with adenine, and the other, which is due to the moving of electrons within that nucleotide’s sequence it called the enol form.
-In the enol form, the thiamine makes h- bonds with guanine, and guanine in the new strand will make bonds with cytosine, so instead of having adenine in the new strand which replaces the thiamine we will have cytosine, this results in a change in the nucleotide's sequence of the gene.
-so spontaneous mutations are due to tautomerization which is the result of a change in the physical state of the nucleotide as a consequence of instability of moving electrons that permit such erroneous h-bonding .

-Mutation can be induced in vitro, here are many mutations that allow many processes to take place and there are substances that can promote tautomerization (promote erroneous h- bonding) either due to changing the physical nature of the nucleotide or they also can cause other effects between different adjacent nucleotides or in the backbone of the DNA (breakage of the sugar phosphate backbones of the DNA).


**Mutagens

- A mutagen is a natural or human-made agent (physical or chemical) which can alter the structure or sequence of DNA. >>> induce and promote mutation.
- We can test physical or chemical agents in vitro by a test known as" the aims test " this test was developed by a scientist named aims to examine the mutagenisty of substances ( to know if this substance can cause mutation or cancer ) because all carcinogens are mutagens
but not all mutagens are carcinogens ,but 85% of mutagens are carcinogens. However all those substances that can cause cancer can cause mutation.

-there are chemical and physical agents that cause mutation …
Chemical agent that causes mutations:
* 5-Bromouracil: analogous to thiamine but it binds to G instead of A , it is similar in structure to T ,but if it is incorporated in DNA -after adding it to a medium where a bacteria grow-it will binds to G instead of A binding to G, so it will lead to a mutation.
*(2-aminopurine): analogue of adenine but it binds to C so mutation
takes place too.

*Nitrous acid (HNO2) causes deamination of nucleotides and deaminated nucleotides make wrong h- bonds, this has the same effect of heat.
Both nitrous acid and heat cause nucleotides deamination promoting erroneouse h- bonding

*Akylating agents (eg ethyl-methyl sulfonate) can induce changes they alkylate nucleotides, and alkylated nucleotides (addition of an alkyle group) will promote erroneous h- bonding.

*Acridine dyes ( eg. proflavin) cause insertion or deletion of nucleotides leading to a frame-shift mutation , so we can induce this kind of mutation by the addition of these substances ( important especially in food).

Physical agents that cause mutations:

1_heat: causes deamination of nucleotides as nitric acid.

2_UV light: it can be used as germ side because it can cause pyrimidine (thymine) dimmers. i.e it connects two adjacent thymines at the same strand .

Note: During replication (copying) of DNA each nucleotide is copied at one point of time so it should be presented as a single nucleotide.

-So if two nucleotides are connected together by covalent bonds they will not be copied and the new strand will contain no nucleotide in the new strand opposite to the template because there are two thymine that are connected to each other because of exposure to UV light .this change in the nucleotide's sequence will cause a mutation .
UV light can also cause hydrated pyrimidines, addition of water molecule to thymine which also leads to an erroneous strand that doesn’t contain a nucleotide that is facing or in place of that hydrated pyrimidine.

So UV light kills bacteria by causing mutations, and these mutations can result in the death of the bacteria.

3_Gamma and x-rays: are ionizing type of radiations, they ionize water which creates hydroxyl radicals that induce breakings in the backbone of the DNA and if DNA breaks it can't act as a template for the synthesize of new nucleotides, so this leads to a mutation in
the new strand.

-Now if we examine the ability of a substance to induce mutation this will be a good test to a assess it's cancerogenesity, that is done by using a bacteria that in called " salmila tayphomeria " the gene of this bacteria cell has hot spots ( i.e if it is exposed it to a mutation it can undergo mutation ) this organism can't grow unless provided with histadine; because it has a mutation that doesn't allow her to synthesize histadine, so if there is no histadine in the medium this bacteria will not grow .

-To test the ability of a substance to induce mutation in this organism we culture the organism with the substance with a chemical, if it grows in a medium that is lacking histadine that means that the mutation had take place,which allows the organism to mutate and synthesize histadine .the growth of the organism indicates that the mutation had take place due to the exposure to the substance that is being tested .

-This is a rough method for testing substances there are many more accurate methods that are used. And it’s also a screening method, i.e if we want to examine a large quantity it can't make sequencing of the gene. And as we know sequencing of the gene is the only way to
Determine if a mutation has taken place or not .There are some machines that can give very accurate sequencing of the gene.


**Gene transfer:

-The second source of biological variation is gene transfer (transfer of genetic material).
-In bacteria this is very common .and there are more than one mechanism for gene to transfer and since the gene is transferred, this indicates that’s there is a donor and a recipient. Usually these organisms should be related, so that recombination can take place.
the segment which is transferred can recombine with the gene (genetic material ) of the donor's recipient cell , but if they are unrelated this DNA usually is degraded and won't become part of the genetic material of the recipient's cell, so donor and recipient
should have a certain degree of relatedness then recombination can take place .

- Now DNA homology is required and usually part of the genome passes from the donor to the recipient’s cell.
-The mechanisms that are involved are in order of importance and in order of frequency, as:
transformation ( meaning that transformation is less important than others and it's less common ) , transduction is more common and important than transformation, conjugation which is the most important mechanism of transfer of the genetic material and the most common .
- Transformation is not important because it's believed to be less significant in variation that took place among bacteria. Bacteria varies and they continue to vary, and one of the sources of variations is the transferred genetic material.
Conjugation is more important and common than transduction, but the least important and least common is transformation.

Now we will discuss each mechanism in details:
- Transformation: by definition is the uptake of the soluble DNA from the environment.
As u know bacteria dye and after dying it disintegrate and they release their genetic material in the environment ,so if another related bacteria is grown in the same environment it can theoretically uptake the DNA which is present as a soluble material in the
Environment, however, you know that the DNA has high molecular weight and its difficult for the organisms to uptake it. So the process is limited and restricted to a few bacteria such as:
Bacillus subtilis
Streptococcus pneumonia
Hemophilus influenzae
Neisseria gonorrhea
E.coli

-In the presence of high concentration of calcium salt and high temperature.
-Those are the few organisms from millions of bacteria that are capable of up taking soluble DNA which is existed in the environment freely and this process is known as transformation ,and that’s why transformation is not believe to have contributory in variation, first
of all its difficult to perform it and its restricted to a few organisms (not common) and usually these organisms change certain feature factors as a consequence of transformation .

The factors that affect the process of transformation include:
1_DNA size and state weather small or large, small -DNA can be taken easily, and single strand can betaken more efficiently than double stranded DNA.
2_ The presence of a factor know as "competent factor of the recipient "The previous mentioned recipient kinds of bacteria has to synthesize a factor that enables them to uptake DNA so this factor is required for transformation, if this factor isn't
Present, bacteria can't uptake soluble DNA.

-The steps that are involved in transformation start with the uptake of DNA then if it is homologous to the DNA that is present in the bacteria recombination takes place, so
recombination process depends on relatedness between donor and recipient DNA .
-Transformation was the first process to take place.
- Scientific “Greffith” described transformation in 1928, before the DNA was even discovered, he describes it in on of these organisms that was mentioned previously (streptococcus pneumonia).
-Streptococcus pneumonia are organisms that are either rough orsmooth ) capsuled or lack a capsule ) and this capsule as we know is made because of an enzymes that synthesize the capsule , they are either fructosyltransferaseas or glucosyltransferases ( either
glucose or fructose ) and these enzymes are proteins .
-He noticed that if he heat killed smooth organisms and mix them with rough organisms those rough organisms become smooth, the smooth were killed, so they will not be able to grow, so the growth of the smooth organisms indicates that the rough were transformed into smooth organisms. that's why they named it transformation, then he went further and he next heat killed smooth with viable rough and inject them into a mice, then the mice died because of infection pneumonia that developed in mice and he smooth organisms from those animals .He concluded that, a substance as passed from the killed smooth to
the rough converting them to smooth and that’s why he called this process transformation and it was impossible for him to determine the DNA . But he demonstrated the process of the transformation.

-The second process of genetic transfer is known as transduction which is the transfer of the genetic material from one bacterial cell to the other via a virus.
- Viruses are simple structures composed of a nucleic acid (DNA or RNA) core and a protein coat which surrounds the genetic material.
- Viruses can infect all organisms (humans, animals, bacteria) even viruses can be infected by viruses.
-Those viruses that infect bacteria are known as "bacteriophages" because they can lyse (phage the bacteria), now when a virus infect a bacteria it attaches to the bacteria cell via fibers.
-The virus has a head, tale and fibers, the head is made of protein and contains the nucleic acid (DNA or RNA) so the bactriophage (the virus) binds to the bacteria cell via its fibers
and infects it's genetic material in the bacterial cell .
-So what enters is the genetic material of the virus, if it’s a DNA virus this DNA will redirect the whole machinery of the bacterial cell to produce viruses.

There are two types of life cycle for bacteriophags:
1- letic .
2- lysogenic (template).

-After the entrance of the virus into the bacterial cell it synthesizes its DNA, its protein, lyse the bacteria and leave it, so there in nothing to harm of transferring the genetic material
, the virus kills the bacterial cell.

-But in certain occasions for certain viruses the genetic material of the virus become integrated in the genome of the bacterial cell (it doesn't lyse it) it becomes part of the bacterial cell, so while the bacterial cell replicate they replicate the genetic material of the virus and become part of the bacterial cell, but it has the potential to cause lyses of the bacterial cell.

-This DNA has the potential to shift from a lysogenic to a letic cycle. That’s why its called lysogenic or template. So non-letic is potentially letic that’s why they are lysogenic. So it continues to replicate and millions of bacteria will be produced carrying this genetic material

-Now if the bacterial cell is exposed to a high temperature or to UV light the integrated viral DNA can be activated to lyse. And once it lyses, it forms new viruses that will leave the cell. viral DNA that is present as part of the bacterial genome takes a segment of the bacterial genome with it, during replication it doesn't leave as a viral DNA only but it takes part of the
bacterial cell with it .

-If it takes only adjacent segment of the DNA with it, this type of transduction will be called restricted (specific type of transduction), meaning its restricted to the five adjacent to the
integration site for the viral DNA .

-But in other conditions the virus has a roughly equal chance to take any part of the bacterial cell genome with it, and this is called the generalized restriction.

-SO the virus enters to the bacterial cell and become part of the genome, replicate as part of the genome, but at once time it decides to leave the bacterial cell, once it leaves the bacterial cell it takes DNA from the bacterial cell with it , that DNA could only the segment adjacent to the site of the integration OR it could affect any part of the DNA which is called generalized transduction as opposed to the restricted transduction.

-The new virus carrying part of the bacterial genome with it can infect new bacterial cell and it will transfer the genetic material from that bacterial cell into the new bacterial cell and that’s why a new characteristic that is not present in this bacteria will appear in the future generations.

-The best example is defteria bacteria which is type of organisms that is characterized by having the ability to produce a powerful toxic, which transferred from one bacterial cell to the other by a virus known as the "tempretphage for the defteroids" or "lysogeneic
bacteriophage for defteria" .

-So a bacterial cell alonging to the coroniom baterial species (eg defteria ) will acquire the property of toxic production if infected by a virus carrying the gene coding for the toxic production …that’s how bacteria converted from non-toxigenic into toxigenic .
The same whole process for scalet fever caused by streptococcae, this due to a toxin called "erthrogenic toxin”.

-The gene coding for the production of erthrogenic toxin is transferred from one bacterial cell to the other by viruses >>>process of transduction.

-So transduction is the process of transfer of the genetic material between bacteria by viruses which are abundant in nature and can transfer certain characteristics and properties between bacteria.


Done by: Isra’a Drarjeh
L. # 5
Date of L. 26/9/2010
The 2sd L.