DECEMBER 16, 2014; TUESDAY; at 2.2; 9.30 A.M. - 12.30 P.M.
Hello! And welcome to the beginning of what is sure to be an interesting last week summer course!
Ha. Recently done my adopted microbe project which is
Helicobacter pylori is my adopted microbe. I used cliptomize application to make this digital scrapbook. It was really help my study. Thank to Him a lot. Check it out!
The lecture start by continuing topic on Microbial Growth that we hold last week:
Isolation/Culturing Maintaining and Preserving Microbes
This topic is important for the next semester during lab session.
What I have learnt is about:
1.
Aseptic technique which is carrying out a procedure under controlled conditions in a manner that will minimize the chance of contamination.
For example : While making Tapai, there are some precautions. Firstly, the person should take a shower before make Tapai. Those who in menstruation are not allowed to make Tapai to avoid the Tapai becomes sour and in red colour. OMG! haha.. Then, wash all the apparatus or tools with hot water may be to kill the bacteria. For better in making Tapai, let nobody know when you want to make Tapai and make it alone. More people, talk more. It is bad because contamination will occur. Bacteria is found everywhere included our mouth. So, I hope you guys get the point what I trying to say.. :D
Steps in aseptic technique:
1. Clear the bench with alcohol (e.g. Detol)
2. Take an agar/medium
3.Take sample
What is suitable medium that we want to use?
= It is depends on what culture we want to isolate.
Types of Culture Media
1. Chemically defined media - The exact chemical composition isknown
2. Complex media - Exact chemical composition is not known
Culturing Microorganisms
1. Liquid culture (mostly those with flagella)
2. Culture plates
We culturing the bacteria to obtain pure culture and to use suitable medium.
Pure culture is a population of identical cells originating from a single cell and obtained by working in aseptic environments. Bacteria is divide by binary fission.
When we take a sample from soil which is the most abundance of bacteria (over growth), we have to dilute the sample.
Some cases, the grown culture is a mix culture. They have to transfer bacteria by bacteria into sub-culture. Bacteria is move to favourable substance (called chemotaxis)
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| Chemotaxis, Haptotaxis, Necrotaxis |
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DECEMBER 18, 2014; THURSDAY; at 1.4; 11.00 A.M. - 2.00 P.M.
After a quick lunch, it was off to the class, and a quick lesson on microbial growth s by Dr. Wan. In this class session, what I have learnt is about:1. Anaerobic culture methods. It needs anaerobic environments that including
- reducing compounds (e.g Thioglycholate, Cystein, anything with -SH, indicator (resazurin). Resazurin is a blue dye, it is used as an oxidation-reduction indicator in cell viability assays for bacteria and mammalian cells, and for measuring aerobic respiration and exchange with the hyporheic zone in streams. Usually it is available commercially as the sodium salt.Resazurin (pH indicator below pH 3.8 above pH 6.5)
- Anaerobic jar that impermeable to Oxygen
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| anaerobic jar |
- Catalyst (e.g platinum or palladium) in Lid or on Gas Pack
Gas Pack
Anaerobic chamber
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| anaerobic chamber |
- Deoxygenerator
Some of procedure in this method are using test tubes that contained melted agar is because to prevent entering of oxygen. The test tube will be rolling in ice to forms thin layer of agar that freeze on test tube wall.
Other technique to provide rich anaerobic environment:
1. Oxyrase
2. Avoid the need for more cumbersome apparatus
Culture Media : A nutrient material prepared for the growth of microorganisms in a laboratory.
Requirements:
1. Must be sterile
2. Contain appropriate nutrients
3. Must be incubated at appropriate temperature
Culture: Microbes that grow and multiply in or on a culture medium.
If we change the chemical culture, the culture cannot grow.
Some info, cellulose as a substrate to analyze cellulase assay that using three substrate to complete a cyclic.
Four types of agar plate demonstrating differential growth depending on bacterial metabolism.
Blood Agar : To cultivate all the fastidious microorganisms.
Eosin Methylene Blue (EMB) Agar :
Macconkey Agar :
Honestly, I dont really clear about the differences by using different type of culture media based on the pictures.
Continue with new sub-sub-topic on
1. Bacterial Growth.
Bacterial growth is all about an increasing number of cells may be via binary fission for some bacteria and some grow by budding, spores (actinomycetes), fragmentations.
2. Population Growth
Is a generation time or doubling time which is depends on environmental factors for each cell to divide or a population to divide.
3. Exponential Growth
We have to know an take serious about the calculation which is very important.
Generation time = 60 minute x hours
Number of generations
The generation time is the time interval required for the cells (or population) to divide.
No of
generations = log10 no. of cells (end) – log10 no. of
cells (beginning)
0.301
We just done an assignment on this topic and some of the questions edited is :
You fall down and scrape your knee and
get infected with 5 Streptococcus pyogenes cells. After 4 hours, without
medical intervention, how many bacteria will be ravaging your body? Let’s say
that for every 1 million bacteria, a centimeter of flesh is consumed. After 4
hours, how much tissue would be lost? Are you still alive and would you want to
be? (This problem is not fact based.)
=Still
alive but need to amputate because of gangrene.
Gangrene is a potentially life-threatening condition that arises when a considerable mass of body tissue dies (necrosis). This may occur after an injury or infection, or in people suffering from any chronic health problem affecting blood circulation. The primary cause of gangrene is reduced blood supply to the affected tissues, which results in cell death. Diabetes and long-term smoking increase the risk of suffering from gangrene.
There are different types of gangrene with different symptoms, such as dry gangerene, wet gangrene, gas gangrene, internal gangrene and necrotizing fasciitis.
Gangrene is caused by a critically insufficient blood supply (peripheral vascular disease) or infection. It is associated with diabetes and long-term tobacco smoking. This condition is most common in the lower extrimities.
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| dry gangrene |
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| gas gangrene |
So scaryyy !!
After we took the culture from meida, we have to do
gram staining to know their characteristics. Then, will do analysis and make a growth curve.
Four characteristic phases of the growth cycle are recognized.
1. Lag Phase.
Immediately after inoculation of the cells into fresh medium, the population remains temporarily unchanged. Although there is no apparent cell division occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity.
The length of the lag phase is apparently dependent on a wide variety of factors including the size of the inoculum; time necessary to recover from physical damage or shock in the transfer; time required for synthesis of essential coenzymes or division factors; and time required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium.
2. Exponential (log) Phase.
The exponential phase of growth is a pattern of balanced growth wherein all the cells are dividing regularly by binary fission, and are growing by geometric progression. The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation. The rate of exponential growth of a bacterial culture is expressed asgeneration time, also the doubling time of the bacterial population.
3. Stationary Phase.
Exponential growth cannot be continued forever in a batch culture. Population growth is limited by one of three factors:
i. exhaustion of available nutrients;
ii. accumulation of inhibitory metabolites or end products;
iii. exhaustion of space, in this case called a lack of "biological space".
During the stationary phase, if viable cells are being counted, it cannot be determined whether some cells are dying and an equal number of cells are dividing, or the population of cells has simply stopped growing and dividing. The stationary phase, like the lag phase, is not necessarily a period of quiescence. Bacteria that produce secondary metabolites, such as antibiotics, do so during the stationary phase of the growth cycle (Secondary metabolites are defined as metabolites produced after the active stage of growth). It is during the stationary phase that spore-forming bacteria have to induce or unmask the activity of dozens of genes that may be involved in sporulation process.
4. Death Phase/logarithamic decline phase.
If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines. (Note, if counting by turbidimetric measurements or microscopic counts, the death phase cannot be observed.). During the death phase, the number of viable cells decreases geometrically (exponentially), essentially the reverse of growth during the log phase.
Direct Methods of Measurement
1. Plate count (continued):
A. Pour Plate:
- Introduce an inoculum into an empty Petri dish.
- Disadvantages:
• Not useful for heat sensitive organisms.
• Colonies appear under agar surface.
B. Spread Plate:
- Introduce an inoculum onto the surface of Petri dish.
- Advantages: Colonies will be on surface and not exposed to melted agar.
2. Filtration: Used to measure small quantities of bacteria.
• Example: Fecal bacteria in a lake or in ocean water.
- A large sample is filtered to retain bacteria.
- Filter is transferred onto a Petri dish.
- Incubate and count colonies.
3. Direct Microscopic Count :
- A specific volume of a bacterial suspension is placed on a microscope slide with a special grid.
- Stain is added to visualize bacteria.
- Cells are counted and multiplied by a 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)
Number of bacteria = no of cells counted
volume of area counted
Indirect Methods of Measurement
1. Turbidity:
- As bacteria multiply in media, it becomes turbid.
- Use a spectrophotometer to determine % transmission or
absorbance.
- Multiply by a factor to determine concentration.
Advantages:
• No incubation time required.
Disadvantages:
• Cannot distinguish between live and dead bacteria.
• Requires a high concentration of bacteria
2. Periodic transfer of strains to fresh medium
3. Incubate and store
in the refigerator (viable for a few weeks)
Preserving Bacteria Culture:
1. Short-term storage [refrigeration]
2. Long-period storage
:: [Deep freezing]
temperature range (-50 to -95)
(-195 degree celcius)
Storage under liquid nitrogen (-196)
:: [Lyophilisation (freeze-drying) ]
temperature range (-54 to -72)
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DECEMBER 19, 2014; FRIDAY; at 2.2; 8.00 A.M. - 12.00 P.M.
Last tutor session for this semester1.
Microbial Control Methods is devided into physical agents, chemical agents, mechanical removal methods and biological agents.
Physical agents : Use external factors from the environment (e.g heat, radiation)
Chemical agents : Using apparatus (e.g gases, liquids)
Mechanical removal : Filtration of air or liquid
Biological agents : Use other organisms (Predator, Virus, Toxin)
Frequently Used Terms
1. Sterilization : The complete removal or destruction of all viable microorganisms, used on inanimate objects.
2. Commercial sterilization : KIllinbg C.botullinum endospores
3. Disinfection : The
distruction or removal of vegetative pathogens but not baterial endospores (usually on inanimate objects)
inanimate = lifeless
4. Antisepsis : Chemicals applied to body surfaces to destroy or inhibit vegetative pathogens.
5. Chemotheraphy : Chemicals used internally to kill or inhibit(stop) growth of microorganisms within host tissues.
6. Demerging : Removal of microbes from a limited area.
7. Sanitization : Lower microbial counts oneating utensils.
8. Biocide/Germicide : Kills microbes
9. Bacteriostasis: Inhibiting, very longer time to kill copared to biocides to kill microbes.
10. disinfectants :Agents usually chemical used for disinfection used on inanimate objects
11. Antiseptics: Chemical agents that kill or inhibit growth of microorganisms when applied to tissue.
- Antimicrobial agents
-Chemotheraphy
-agents that kill microorganisms (cidal agents) or inhibit their growth (statistic agents) -die gradually.
The pattern of microbial Death
- Occurs exponentially
- Not killed instantly
- VBNC (Viable but not nonculturable condition)
The pattern depends on
- Number of microbes
- Duration of exposure
- Microbial characteristics (e.g. resistance to high temperature/descication) -->endospore:cell that can resistance most condition.
- Concentration or intensity of an antimicrobial agent
- Population (one sp. in habitat) composition
- Temperature
- Local environment
# LD 50 --> little dose amount of antibiotice that kill 50% of microbes/organism
Microbial agents do alter of membrane permeability, damage to proteins, and damae to nucleic acids that impact to DNA replication, production of amino acids, cell wall and act.
1. Heat
- TDP : Lowest temperature at which all cells in a culture are killed in 10 min
- TDT : Time to kill all cells in a culture
- DRT : Time in minutes to kill 90% cells at a given temp.
-Moist heat can denatures protein
- Autoclave : Steam under pressure at temperature: 121 degree cel (15 minutes) with pressure 5 psi.
- Moist heat actually can destroys bacteria, viruses, and fungi but not spores (cannotbe sterilize). It will degrades nucleic acids, denatures proteins, and distrupts membranes. For becteria, it is only for mesophilcs.
2. Filtration
3. Radiation
How do we know food spoilage?
We can indicate through the odour of food, taste, and texture.(organo leptic). Food borne pathogen that cause disease from food.
How do we control growth of microbes?
Through preservation with low temperature that inhibits microbial growth.
- Refrigertion
- Deep freezing
- Lyophilization
If using high pressure lead to denatures proteins. Desiccation prevents metabolism while osmotic pressure causes plasmolysis.
When we talk about filtration, of course we think of water filter.
Usually, the size of filtrate that can pass through the filter are <0.01nm.
The important thing that we should know is about things that cannot be sterilize by using autoclaves.
1. Buffer
2. Antibiotic solution (e.g penicilin)
We learn about chemical agents which we use-dilution test or diffusion method (in Laminar flow)
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| Evaluating a disinfectant |
The properties of the disinfectant – how it works determines what it will be effective against.
Concentration of the disinfectant - requires proper hydration.
The presence of organic matter can act as a buffer
The degree of contact with microorganisms – if the surface needs cleaning or is porous microorganisms can escape contact with the disinfectant.
Temperature should also be considered, since increased temperatures usually enhance the efficacy of disinfectants.
Time left in contact is also an important consideration.
In the use-dilution test, bacterial (S. choleraesuis, S. aureus, and P. aeruginosa) survival in the manufacturer’s recommended dilution of a disinfectant is determined.
Viruses, endospore-forming bacteria, mycobacteria, and fungi can
also be used in the use-dilution test.
In the disk-diffusion method, a disk of filter paper is soaked with a chemical and placed on an inoculated agar plate; a clear zone of inhibition indicates effectiveness.
Mycobacteria resistance to antimicrobial agents because of its cell wall that contains endospores. Vampirelics is a group of protozoa that kill fungus
Protozoa eat bacteria.
Next, we ae going through topic on Antimicrobial Chemotheraphy.
Chemotherapy means to destroy with chemicals. Chemotherapeutic agents will kill rapidly dividing cells (like bacteria!).
Antimicrobial means it kills or inhibits microorganisms, like bacteria (fungi, protozoans, etc).
It's less harmful to the host (in this case, you) because it'll kill the bacteria but not the cell.
When you see long words like this try to break them down. Oftentimes the definition is hidden in the question.
Anti = against
Microbial = microbes (tiny organisms).
So, antimicrobial must mean that it is against microorganisms, either killing or impairing.
Chemo = chemicals.
Chemotherapy = therapy using chemicals.
If we put two and two together we get a chemical compound that kills bacteria.
This might help me to know more about antimicrobial chemotheraphy more
(http://www.microbiologybytes.com/iandi/9a.html)
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| Paul Ehrlich (1904) |
Paul Ehrlic
- developed concept of selective toxicity
- identified die that effectively reated African sleeping sickness (Trypanosoma gambiense)
- More details about this man? What he do? http://www.paul-ehrlich.de/Research/research.htm
Sahachiro Hata
- Working with Ehrlic, identified asenic compounds that effectively treated syphilis.
Therese Trefouel
- discovered sulfonamides and sulfa drugs.
.
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| Penicilin notatum |
Bacteremia is the presence of bacteria in the blood.
General Caracteristics of Antimicrobial Drugs
1. Selective toxicity
2. Therapeutic dose
3. Toxic dose
4. Therapheutic index
5. Side effects
Antimicrobial agents are the most valuable means available for treating bacterial infections. However, the administration of therapeutic doses of antimicrobial agents to patients is a leading cause of disturbance of the normal gastrointestinal microflora. This disturbance results in diminishing the natural defense mechanisms provided by the colonic microbial ecosystem, making the host vulnerable to infection by commensal microorganisms or nosocomial pathogens. In this minireview, the impacts of antimicrobials, individually and in combinations, on the human colonic microflora are discussed.
6. Narrow-spectrum drugs
7. Broad-spectrum drugs
8. Cidal agents
9. Static agents
10. Effects of an agent may vary
11. Effectiveness expressed in two ways:
i. MIC - Minimal
inhibitory concentration
ii. MLC - Minimal
lethal concentration
Kirby-Bauer Method
Check out this video (
http://www.youtube.com/watch?v=sx1uDYSfINA)
Ha. This is my last assignment of BMY3101. I have difficulties through out this video. The original one that I made at evening cannot open. May be due to viruses from external camera. The file is corrupted and cannot be repair. So, have to made a new video in last minute.
Before that, this is my first video in the first week being a microbiology student. :D
Thats all about what I have learn in Microbiology for this semester. I will miss updating this blog nightly with images and updates from the course to give insight into the happenings in the class, out in the field, and wherever else we venture out to. About to cry right now.
I'm gonna miss every moment that we spend together, :'(
Dear friends, wish you luck for final and to my lecturer Dr Wan Zuhainis thank you so much for teach and motivate us through out 14 weeks. Heart you :*
Byee~
