براءة حب
๑ . . عضو متميز . . ๑
- التسجيل
- 3 يوليو 2004
- رقم العضوية
- 1952
- المشاركات
- 507
- مستوى التفاعل
- 12
- الجنس
Microscope
Microscopes are devices by which one can make visible details of structure too minute to be seen by the unaided eye, or too poorly differentiated from their surroundings to be identified."
Leeuwenhoek was a man with many talents, his most important attributes were creativity, power of observation, and ingenuity. Leeuwenhoek was a common man without any fortune or formal education, so he had to work for a living. Leeuwenhoek made simple (one lens) microscopes. He was not the first person to build a microscope, but the microscopes that he did build were the best ones for that time period. Leeuwenhoek was the first person to describe bacteria (from teeth scrapings), protozoans (from pond water), helped to prove the theory of blood circulation. He gained much of his inspiration form reading Hooke's Micrographia.
What are Bacteria?
Until recently, the term bacteria was used for all microscopic prokaryotes. But, it turns out that there are two groups of prokaryotes that differ from each other in just about every way except size and lack of a nucleus.
These are now distinguished as the:
• Bacteria; the "true" bacteria (also known as Eubacteria)
• Archaea; (also known as Archaebacteria)
The archaea are so different from the bacteria that they must have had a long, independent evolutionary history since close to the dawn of life. In fact, there is considerable evidence that you are more closely related to the archaea than they are to the bacteria!
Properties of Bacteria
• prokaryotic (no membrane-enclosed nucleus)
• no mitochondria or chloroplasts
• a single chromosome
o a closed circle of double-stranded DNA
o with no associated histones
• If flagella are present, they are made of a single filament of the protein flagellin; there are none of the "9+2" tubulin-containing microtubules of the eukaryotes.
• ribosomes differ in their structure from those of eukaryotes
• have a rigid cell wall made of peptidoglycan.
• The plasma membrane is a phospholipid bilayer but contains no cholesterol or other steroids.
• no mitosis
• mostly asexual reproduction
• any sexual reproduction very different from that of eukaryotes; no meiosis
• Many bacteria form a single spore when their food supply runs low. Most of the water is removed from the spore and metabolism ceases. Spores are so resistant to adverse conditions of dryness and temperature that they may remain viable even after 50 years of dormancy
Classification of Bacteria
Until recently classification has done on the basis of such traits as:
• shape
o bacilli: rod-shaped
o cocci: spherical
o spirilla: curved walls
• ability to form spores
• method of energy production (glycolysis for anaerobes, cellular respiration for aerobes
• nutritional requirements
• reaction to the Gram stain.
The Gram stain is named after the 19th century Danish bacteriologist who developed it.
• The bacterial cells are first stained with a purple dye called crystal violet.
• Then the preparation is treated with alcohol or acetone.
• This washes the stain out of gram-negative cells.
• To see them now requires the use of a counterstain of a different color (e.g., the pink of safranin).
• Bacteria that are not decolorized by the alcohol/acetone wash are gram-positive.
Although the Gram stain might seem an arbitrary criterion to use in bacterial taxonomy, it does, in fact, distinguish between two fundamentally different kinds of bacterial cell walls and reflects a natural division among the bacteria.
More recently, genome sequencing, especially of their 16S ribosomal RNA (rRNA), has provided new insights into the evolutionary relationships among the bacteria.
Media
Type of media used in lab:
1- Blood Agar (BA)
Enrich media and differential media for hemolytic organisms.
2- Chocolate Agar
Enrich media used for cultivation of pathogenic Neisseria and H. influenza.
3-MacConkey Agar(Mc A)
selective media that differentiate between Gram negative bacteria while inhibiting the growth of most Gram positive bacteria. The medium also differentiates between lactose-fermenting coliforms and lactose nonfermenters, which include potential pathogens.
4- DCA (Deoxy cholate citrate agar)
A good selective media for salmonella and shigella.
5- XLD (Xylose Lysine Deoxy cholate Agar)
6- CLED media
Used to isolate the organisms in urinary tract.
7-Nutrient Agar (NA)
Simple media used in cultivation of different organisms.
Microbiology Media Preparation
Bacteria and fungi are grown on or in microbiological media of various types. The medium that is used to culture the microorganism depends on the microorganism that one is trying to isolate or identify. Different nutrients may be added to the medium, making it higher in protein or in sugar. Various pH indicators are often added for differentiation of microbes based on their biochemical reactions: the indicators may turn one color when slightly acidic, another color when slightly basic. Other added ingredients may be growth factors, NaCl, and pH buffers which keep the medium from straying too far from neutral as the microbes metabolize.
In this exercise, your table will make all-purpose media called nutrient broth and nutrient agar. These 2 media----one a liquid and the other a solid---are the exact same formula save for the addition of agar agar (really---agar agar), an extract from the cell walls of red algae. This particular medium will grow MOST bacteria that we use in lab: however, it is classified as a rather minimal medium since there is no added sugar to it (like trypticase soy agar or plate count agar).
The old way to make media was by the cookbook method----adding every ingredient bit by bit. The only time that is done today is when making a special medium to grow a certain finicky organism, where particular growth factors, nutrients, vitamins, and so on, have to be added in certain amounts. This medium is called a chemically defined medium (synthetic). Fortunately, the most common bacteria that we want to grow will do nicely with media that we commonly use in lab. Some of our media is bought, but most is produced in the prep area behind the lab. Since this type of medium has some unknown ingredients, or sometimes unknown quantities, it is called complex media.
STERILIZATION AND THE AUTOCLAVE
When microbiological media has been made, it still has to be sterilized because of microbial contamination from air, glassware, hands, etc. Within a few hours there will be thousands of bacteria reproducing in the media so it has to be sterilized quickly before the microbes start using the nutrients up. The sterilization process is a 100% kill, and guarantees that the medium will stay sterile UNLESS exposed to contaminants by less that adequate aseptic technique to exposure to air.
Media sterilization is carried out with the autoclave, basically a huge steam cooker. Steam enters into a jacket surrounding the chamber. When the pressure from the steam is at a certain point in the jacket, a valve allows the steam to enter the chamber. The pressure will go up over 15 pounds per square inch (psi): at this point the timer begins to count down---usually for 15 minutes, depending on the type of media. The high pressure in a closed container allows the temperature to go above the highest temperature one could get by just boiling, around 121 degrees C. Therefore, the parameters for sterilization with an autoclave are 121 C at >15 psi for 15 minutes. Fifteen minutes is the thermal death time for most organisms (except some really hardy sporeformers).
The prepared media is distributed in different ways, depending on the form one is making. Broths and agar deeps are aliquotted into tubes and then sterilized. Agar slant tubes are sterilized and then the rack is tilted to allow the agar to solidify in a slanted fashion. Agar medium which will be poured into plates is sterilized in a flask, and then poured afterward. Not all media or solutions can be sterilized via an autoclave. Certain high-protein solutions such as urea, vaccines, and serum will denature in the extreme heat, and so they may have to be filter-sterilized without heat.
Patient Samples
After preparation of the media. We saw the patient samples that awarded to the lab and also how to make the culture for this samples. There are many type of samples:
Faeces (F), High Vaginal swab (G), Semen culture (Gs), Urethral for Gs, Throat (Th), Pus (P), Urinary catheter (Uc), eye (Ey), Ear (E) and placental (Pl).
Streaking the plates and incubation
The streak plate technique is the most widely used method of obtaining isolated colonies from a mix of cultures. The streak plate technique is essentially a method to dilute the number of organisms, decreasing the density. This allows for individual colonies to be isolated from other colonies. Each colony is considered "pure," since theoretically, the colony began with an individual cell.
Actually most of the samples were cultivated on the BA, chocolate Agar and McA. There are to selective media just for the stool samples : DCA and XLD media.
After streaking the plates (BA, McA and chocolate Agar) with the patient samples. Incubation were done for 24hr. After 24hr plates were checked for the presence of any pathogenic bacteria (for example the pathogenic bacteria on the BA produce clear zone around the colony due to the hemolysis of blood) and by comparing the plates of different media we saw the morphology of the bacteria on the different media.
Also we have some problem when we identified the bacteria. Because there are a lot of organisms that have the same appearance and morphology of the colony so we do some stains and tests to know exactly the pathogenic one:
1- Gram Stain
Gram-staining Procedure:
Gram-staining is a four part procedure which uses certain dyes to make a bacterial cell stand out against against its background. The specimen should be mounted and fixed on a slide before you procede to stain it. The reagents you will need to successfully perform this operation are:
• Crystal Violet (the Primary Stain)
• Iodine Solution (the Mordant)
• Decolorizer (ethanol is a good choice)
• Safranin (the Counterstain)
• Water (preferably in a squirt bottle)
Before starting, make sure that all reagents, as well as the squirt-bottle of water, are easily accessible because you won't have time to go get them during the staining procedure. Also, make sure you are doing this near a sink because it can get really messy.
STEP 1: Place your slide on a slide holder or a rack. Flood (cover completely) the entire slide with crystal violet. Let the crystal violet stand for about 60 seconds. When the time has elapsed, wash your slide for 5 seconds with water. The specimen should appear blue-violet when observed with the naked eye.
STEP 2: Now, flood your slide with the iodine solution. Let it stand about a minute as well. When time has expired, rinse the slide with water for 5 seconds and immediately procede to step three. At this point, the specimen should still be blue-violet.
STEP 3: This step involves addition of the decolorizer, ethanol. Step 3 is somewhat subjective because using too much decolorizer could result in a false Gram (-) result. Likewise, not using enough decolorizer may yield a false Gram (+) results. To be safe, add the ethanol dropwise until the blue-violet color is no longer emitted from your specimen. As in the previous steps, rinse with the water for 5 seconds.
STEP 4: The final step involves applying the counterstain, safranin. Flood the slide with the dye as you did in steps 1 and 2. Let this stand for about a minute to allow the bacteria to incorporate the saffranin. Gram positive cells will incorporate little or no counterstain and will remain blue-violet in appearance. Gram negative bacteria, however, take on a pink color and are easily distinguishable from the Gram positives. Again, rinse with water for 5 seconds to remove any excess of dye.
After you have completed steps 1 through 4, you should blot the slide gently with bibulous paper or allow it to air dry before viewing it under the microscope. DO NOT RUB THE SMEAR!
2
-Biochemical test:
- Urease test
In this row of urea broth, each of which has been inoculated with a different G- organism, the only organism which is positive for urea hydrolysis is Proteus vulgaris --the tube which has turned pink, third from the left.
In Saqer hospital microbiology lab I did this test to differentiate between two patient sample that were cultured on XLD Agar. The two plate show the same morphology of the colony (black round colony) that we cannot decide if it is salmonella or proteus.
Materials
- 24- 48 hr nutrient broth cultures of proteus and salmonella.
- Urea broth tube.
-Bensen Burner.
- Test tube rak
- Inoculating loop
- Incubater at 370C.
Procedure:
1- Label each of the urea broth tubes with the name of bacterium to be inoculated (salmonella and proteus), with leaving one tube for control.
2- Using aseptic technique, inoculate each tube with the appropriate bacterium by means of loop inoculation.
3- Incubate the tube for 48 hr at 370C.
Observation:
Formation of red color indicate the positive result.
Comment:
• Some bacteria are able to produce an enzyme called urease that attacks the nitrogen and carbon bond in amide compounds such as urea, forming the end products ammonia, CO2, and water.
• Urease activity is detected by growing bacteria in a medium containing urea and pH indicator such as phenol red. When urea is hydrolyzed, ammonia accumulates in the medium and makes it alkaline. This increase in pH causes the indicator to change from orange- red to deep pink or purplish red and is a positive test for urea hydrolysis. Failure of a deep pink color to develop is a negative test.
- Oxidase test
A positive test for the production of the cytochrome oxidase enzyme--the culture turned purple within 15 seconds of adding the oxidase reagent to the swab.
- Catalase test
The catalase reagent is 3% H2O2 (hydrogen peroxide), which is the strength of store-bought peroxide.
