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UNIVERSI1Y Of JORDAN

Faculty OF Medicine

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LECTURE NO:

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Microbiology

Lect.NO.4

Date: 21/9/2008

Bacterial growth and nutrition Continued • •

Bacteria are divided into two major groups: 1- Heterotrophic: mostly pathogens related to our human body. 2- Autotrophic: widely distributed in nature, and rarely associated with infection. • The term saprophytic bacteria describes bacteria which are found in nature, it is generated through food or water contamination and reach our intestine, but generally with no significant effect. .. Culturing bacteria: it is important to isolate bacteria from an existing specimen like blood, urine, ... Culture growth should be associated with certain growth conditions like neutral, acid, or alkaline ph. For Ex: we have a type of bacteria called Vibrio Cholera, the causative agent of the disease cholera, and this organism despite the fact it can infect our intestine, it can't be well isolated in a neutral medium, we have to use alkaline medium with a ph of 8.5209. • The terms acidophilic, mesophilic, thermophilic bacteria are related to the temp that supports the optimal growth, which is important to recognize the organism in clinical specimen. c Generally the pathogenies are considered the mesophilic bacteria, which means that they grow in a wide range of temp between 20 - 40 degrees, where as the acido, and thermo are less causative agents of diseases. (j

How can we recognize bacterial growth? Look at the slide with the title containing MacConKey • We have two types of media :­ 1- Solid medium: where you can recognize on the surface the presence of some E-coli colonies, this type is called MacConkey agar, this solid medium should be used to recognize the colony, No. of colonies, the morphology of the colony, is it flat or elevated over the surface ... , the color 2-

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Fluid medium: which we call broth, and it can be used exactly as a solid medium to grow organisms capable of living in vitro conditions (out side a living host, as in the lab), we can take samples from food or water, and cultivate them, after incubation of 37 degrees which is the optimal temp for most rapidly growing pathogens, so after 24 hours we can recognize the presence of particles disturbed in this medium known as turbidity (which means the presence of a particle or bacterial cell in a culture medium broth and then the No. of colonies can be exactly estimated) The bacteria uses for replication a very simple method called Binary fission, if we have a single cell in a medium with the appropriate amounts of water and nutrients the cell will be elongated, the cell produces a septum (cross wall), **generation time » the time which is necessary for one single cell to be duplicated. It diffs from one type to the other, it can be for Ex: in relation to Escherichia coli which is found in the intestinal tract, and grows under facultative anaerobic conditions, each generation requires 20 mins, in 24 hr we could have 10/\6 cells, this could be measured by bacterial growth curve. In our body it well usually try to suppress such growth but if the bacteria managed to over come the immune system you can expect in a period of time between 24-72hr to see what the curve in the slide represents. If we put a cell in a fresh medium (1 L of milk for Ex) for cultivation, it will be activated, which means all necessary enzymes will be activated which in turn will induce metabolic activity, this process varies from one kind to the other, it might extend from 1-2 hrs, it is called lag phase

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in length). 2-The sec phase called exponential growth phase (the fastest one), where the No. of cells will witness a logarithmic increase, within 2-3 we will have one million cells. 3-But in this 1L of milk we oniy have a iimited amount of nutrients, so within 10hrs it will utilize all nutrients in this medium and begin to enter the stationary phase, here we have equilibrium between the new viable cells and others which begin to die, because we have certain enzymes between cell wall and cytoplasmic membrane called lysozyme which trigger the process of cell destruction. 4-The last phase happens after 24hrs all needed oxygen, nutrients storages are depleted, and this phase happens slowly it might extend from few days to few weeks. * In the culture medium the system is closed but in our body it is opened with a contentious system (highly regenerative, in­ out), so as long as the infected person is alive the bacteria can utilize more nutrients, and cause damage to all important ,..,. ......... __ ,...

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?Omin~ ~ industry, for Ex: if they want to produce a type of organic acid, VI ~Oll;;:),

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or vitamin, they can do so by using some kind of bacteria and having a continuous culture medium, supplied by oxygen so the bacteria will reach the stationary phase and stay there never reaching the death phase.

Bacteria genetics It is important to understand the role of antimicrobial drugs in the treatment of infection, and how bacteria can resist the various types of antibiotics (with resistance factors), and how they become toxigenic. @

Any bacterial cell carries all genetic Into WhiCh are necessary for growth (production of all necessary enzymes, and the over all structure). All the structural features are encoded in the genetic pole of the bacteria (flagella, capsule, generation time, fimbria ... ), and these genes are arranged !!1 the chromosomes. The bacterial chromosome

is a single stranded DNA, composed of double helix, each gene is composed of a small segment of DNA, that is composed of a small set of nucleotides in the form of triple codon. The No. of nucleotides can be measured by kilo base pairs, base pairs means nucleotides ( A -> T, G -> C). But not all genes are active, some of them are expressed this is called phenotype expression of the bacterial cell, like the color of the colony of the culture medium, it also means the metabolic activity in relation to the utilization of lactose » if the organism is lactose positive (it can break down lactose) this is a phenotype. Where as other genes might be silent, and only under certain conditions they can be activated and expressed. There for, any type of bacteria has two properties: *One called genotype which include all genes in the bacterial chromosomes. **And the other phenotype that include all expressed recognizable characteristics, this can also be recognized in relation to disease, because certain genes can be in the form of invasiveness » produce specific enzymes which affect the mucosa and damage it, or expressed in the production of toxins, and food poisoning like staphylococcus. @

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The central dogma in relation to replication of bacteria can be easily demonstrated like follows » the DNA is replicated to mRNA (transcription), which then is attached to the ribosome, that translate the massage conveyed to specific sequences of amino acids (translation), which are (the amino acids) delivered to the location by tRNA. Bacterial genetics where also introduced in pharmaceutical industry, by capturing a specific gene from humans, like the gene of insulin in the pancreas, and transplanting the gene of insulin into yeast cells, then by allowing them to replicate, they produce insulin in an industrial scale (this process is called bioengineering), also this process was used in producing vaccines for some diseases such as, hepatitis A, B, .... Another topic of great importance to our subject is H(J)'Ifl:f UIiJ! diagn({))se diseases?

if we want to identify the causative agent of a disease, such as pneumonia, we collect a specimen, and then culture it. in the ~

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sequences in the DNA of the clinical specimen, and due to the fact that we have certain conservative regions within the bacterial chromosomes, which are considered as trade marks for each type, then we can cross mach between the obtained ones and the previously obtained and identified ones, this requires a speciai technique known as the peR technique (Polymerase Chain Reaction). IF you have a single cell during the infection (this could happen !! as the immune system greatly reduces the No. of the infecting cells), it will release a few amount of DNA which can't be easily characterized without amplification » to increase the amount of DNA in the clinical specimen (copies).ln short a very powerful technique, it can identify the slightest amount of infectious agents in clinical diagnosis. Ex: we use it in our lab to identify T. B, because the normal T. B culture requires 5 to 6 weeks to know for sure that the person is infected with micro bacterial tuberculosis » (an infectious disease that causes small rounded swellings tubercles to form on mucous membranes, especially a disease pulmonarv tuberculosis that affects the lungs), where as with PCR it takes 5 to 6 hrs, so you can take the necessary steps to isolate the infected person because of the epidemic nature of this disease.

*This is easier for bacteria than for viruses » sometimes we have more than one virus that produce the same clinical picture, like influenza, that is caused by influenza viruses, but it can also be produced by rino viruses, so it is not easy to differentiate between the two. @

As we have said, bacteria contain a single, circular, double stranded, super coiled DNA, which is considered as a single chromosome not separated from the cytoplasm by a nuclear membrane, so it is naked, freely floating within the cytoplasm, not necessarily concentrated in the center of the ceii, the iength of this chromosome is 1200 Micrometer, or roughly 1 Millimeter, so as you can see it is extremely long, but it is super coiled, and it is considered the genome » the main genes of the bacteria, and contain (2 - 5) x 10A 6 nucleotides, and this is enough to contain 1000 to 3000 genes, rarely 5000.

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During bacterial replication whither in nature or in the human body, the DNA strand might lose some nucleotides or, insert (replace) others » Mutation, this can be of two types: 1-Under natural condition it is considered as a spontaneous mutation, and it can't be easily recognized in culture, like E-coli culture, you can't recognize the diff in color, because these changes can be very minimal, like having 10"'7 cells of the same type with 4 to 5 cells with diff characteristics, but if the diff has increased to overwhelming levels, that, it causes a change in the phenotypes of the bacteria, Ex: instead of being susceptible to penicillin, it develops a certain resistance to penicillin. 2-lnduced mutation, for Ex: by ultraviolet, presence of chemicals. These two types of mutations can be controlled by the bacteria to some extent, they contain genes to repair any damage inflected on the DNA sequence also to some extent. *Ex: If we have a bacterial culture of staphylococcus bacterium occurring (staforia ???, the Dr's words) »

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clusters: a bacterium that typically occurs in clusters resembling grapes, normally inhabits the skin and mucous membranes, and may cause disease. These bacteria commonly infect the skin, eyes, and urinary tract, and some produce toxins responsible for septicemia and

If these bacteria acquired a specific virus carrying a gene which is responsible for the production of toxins, the bacteria will produce a strain that produces toxins contaminating our food, and affecting our health. food

poisoning.

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Knowledge is proud that he has leam'd so much; Wisdom is humble that he knows no more. The most intelligent people are the people of doing without, because they love what Allah loves and dislike the world which Allah dislikes. Inexperience is what makes a young man do what an older man says is impossible.

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