- | 04 Apr 2017
Objective: To determine the ability of an organism to grow in 7.5% NaCl and ferment mannitol.
Any significant growth indicates the organism is a Staphylococcus species. The phenol red indicator changes to yellow at low (acid) pH, which is a product of fermentation. Therefore, fermentation of mannitol will change the color of agar to yellow. Orange is negative.
Positive: Growth, yellow color (mannitol "+").
Negative: Growth or no growth; red or orange color (mannitol "-").
Objective: Some pathogens are able to produce exoenzymes called hemolysins which lyse red blood cells and thus their action can be demonstrated on a blood agar plate.
1. Using a sterile loop, inoculate a blood plate (SBA) with the pure culture of the organism to be tested using the quadrant method. Also stab the medium in the second quadrant with your loop. (Some hemolysins show their effects better under lower oxygen concentrations.)
2. Incubate for 48 hours at optimum temperature for the organism.
Interpret by noting the reaction around isolated colonies as follows:
Alpha (α) hemolysis: formation of a green or brown zone around the colonies (due to loss of potassium from the red cells).
Beta (β) hemolysis: complete lysis of cells and reduction of released hemoglobin; a clear zone appears around isolated colonies.
Gamma (γ) hemolysis: no hemolytic reaction (no change of the medium surrounding isolated colonies).
-- The reaction should be checked only around isolated colonies. If you do not have isolated colonies on the blood agar, a lighter inoculation should be streaked and the test repeated.
Objective: DNase mediates the hydrolysis of DNA. Methyl Green indicator is stable at pHs above 7.5 but becomes colorless at lower pHs. The hydrolysis of DNA in the agar by bacterial DNase reduces the agar pH.
1. Using a sterile loop, inoculate a DNA+Methyl Green agar plate with the fresh bacterial culture. Use a heavy streak line for each bacterial strain to be tested. Be sure to label the plate bottom properly for each strain.
2. Incubate at 37°C for 48 hrs.
-- The test is positive if clearing develops around the areas of growth. If the color of the agar around the growth is unchanged, the test is negative (i.e., the organism is not able to produce DNase).
Objective: To determine the ability of an organism to ferment (degrade) a specific carbohydrate in a basal medium producing acid or acid with visible gas. The acid would change the color of the medium in a positive test. The following carbohydrate semi-solid media tubes are available at our lab:
1. Using a sterile needle, stab the tube within 1/4 inch of the bottom with medium inoculation.
2. Incubate for at least 48 hrs. Bacteria that are known to be slow growers should be given up to 96 hours.
Positive: Any yellow color (not orange). It does not necessarily have to be the whole tube. A positive result is referred to as ("+") or (A) or (Acid), as fermentation forms acidic products.
Negative: A red, pink or orange color - no yellow at all.
-- Gas production
Positive: Significant bubbling in semisolid medium (one small bubble is generally negative, caused by the stab). Gas may also cause the medium to get separated from tube. Record as (G) for positive gas production.
Negative: No gas bubbles except those produced by stabbing.
Objective: To determine if the organism is capable of breaking down starch into maltose through the activity of the extra-cellular α-amylase enzyme.
1. Use a sterile swab or a sterile loop to pick a few colonies from your pure culture plate. Streak a starch plate in the form of a line across the width of the plate. Several cultures can be tested on a single agar plate, each represented by a line or the plate may be divided into four quadrants (pie plate) for this purpose.
2. Incubate plate at 37 °C for 48 hours.
3. Add 2-3 drops of 10% iodine solution directly onto the edge of colonies. Wait 10-15 minutes and record the results.
-- Positive test ("+"): The medium will turn dark. However, areas surrounding isolated colonies where starch has been hydrolyzed by amylase will appear clear.
-- Negative test ("-"): The medium will be colored dark, right up to the edge of isolated colonies.
Many different tests have been devised over the years for classification of microorganisms into families, genera, species and even subspecies. Some of these tests are quite simple to perform while others are complicated and may require sophisticated equipment. The tests presented here are among the easier ones that are utilized in major clinical laboratories around the world. These tests are ordered alphabetically in this section. Make sure that you read the complete discussion of each test before you start to perform it.
IMPORTANT: Many of the tests mentioned in the following sections are enzymatic reactions. Therefore to get a correct results, one needs to warm up the culture and all test materials to temperatures between 25-40°C for the reactions to proceed. If you have stored your plates, broth culture or test reagents in the refrigerator, you may need to place them at the 37°C incubator for 15-20 minutes before performing the test.
- AMYLASE TEST (STARCH HYDROLYSIS)
- BILE ESCULIN TEST
- CARBOHYDRATE FERMENTATION TESTS
- CATALASE TEST
- DNASE TEST
- GELATINASE TEST (GELATIN LIQUEFACTION)
- HEMOLYTIC REACTIONS
- MANNITOL SALT AGAR TEST
- MOTILITY TEST
- NITRATASE TEST (NITRATE REDUCTION; DENITRIFICATION)
- NOVOBIOCIN DISC TEST
- OPTOCHIN DISC TEST
- OXIDASE TEST
- OXYGEN REQUIREMENT TEST (Thioglycollate Test)
- PENICILLIN DISC TEST
- SALT TOLERANCE TEST
Objective: To determine the ability of an organism to reduce nitrate to nitrite which is then reduced to free nitrogen gas. The nitrogen in nitrate serves as an electron acceptor. The result of the denitrification process is the production of nitrite:
In this case, all the NO3- will be converted to N2 gas which escapes to the atmosphere. We can test for this step by looking for the absence of NO3- through the addition of Zn powder as described below.
1. Inoculate a nitrate agar slant with your pure culture using a sterile loop to transfer a rather heavy inoculum.
2. Incubate at 37°C for at least 48 hours.
3. Add 2-3 drops of Reagent A and 2-3 drops of Reagent B to your tube. Reagent A is 0.8% sulfanilic acid in 30% acetic acid and Reagent B is 0.6% N,N-dimethyl-α-naphthylamine in 30% acetic acid (CAUTION: Reagent B is a potential carcinogen, so work in the hood and avoid inhaling it or allowing for contact with skin; wash hands thoroughly after work).
Reduction of nitrate to nitrite is indicated if a red color develops quickly (within 1-2 minutes). If no color develops, add a very small amount of zinc powder (~20 mg) to the tube containing the reagents. If a pink to dark red color develops after adding the zinc powder within 5 min., the test is negative (nitrate is present and is not reduced by the organism but zinc has reduced it to nitrite). If no color develops, the test is positive (the organism was able to reduce all the nitrate to nitrite and further to N2 which escaped from the tube).
-- If tubes are stored in the refrigerator, they should first be brought back up to the optimum temperature of the growth condition of the organism.
-- When performing the nitrate reduction test using α-naphthylamine, the color produced in a positive reaction may fade quickly. Interpret results immediately, particularly when performing a number of tests.
-- A strong nitrate-reducing organism may exhibit a brown precipitate immediately after the addition of the reagents. This is due to the effect of excess nitrite upon the p-amino group of the azo-dye and may be reduced by using dimethyl-α-naphthylamine.
-- Some organisms are capable of reducing nitrate to nitrite, yet they destroy the nitrite as fast as it is formed, yielding a false negative result. This nitrite destruction is evident in quite a few bacteria, particularly some Salmonella and Pseudomonas spp. and in Brucella suis.
-- Do not use an excess of zinc; if too much Zn is added, the large amount of hydrogen gas produced may reduce the nitrite (formed from unreduced nitrate) to ammonia (NH3) that could result in a false negative reaction or just a fleeting color reaction.
1. If the plate is refrigerated, it should be allowed to warm up to room temperature and then incubated for 15 min at 37°C before performing the test. Pick a loopful of colonies from a not-too-old pure culture plate and place on a clean glass slide. Do not take your colonies from a blood agar plate. Blood contains catalase; therefore a false positive reaction would be obtained.
2. Add one or two drops of 3% H2O2 and wait 10-15 seconds to observe.
-- Positive test: immediate bubbling (O2 formed).
-- Negative test: no bubbling.
a. When doing the slide test, always add organism to the slide first and then add the reagent since platinum used in the inoculation needle may produce a false positive result. Nichrome wire does not cause bubbling.
b. H2O2 is very unstable when exposed to light. H2O2 decomposition also increases as temperature increases due to dissolved oxygen. Therefore it is important to keep this reagent in the refrigerator at all times when not in use and to shake before it is used.
Objective: To determine the ability of an organism to hydrolyze the glycoside esculin to esculetin and glucose. Esculetin reacts with iron to form a dark brown to black complex. The medium contains 40% bile. Some streptococci that are capable of splitting esculin cannot tolerate an increased concentration of bile. So this is basically two tests: (a) growth on 40% bile and (b) hydrolysis of esculin.
Obtain a bile esculin slant. With a sterile loop, touch a colony of your pure culture to obtain a light inoculum. Uncap the tube and flame the lip of the tube. Insert the loop to the bottom surface of the agar. Touch the agar and gently slide the loop in a zigzag fashion along the surface of the agar as you pull the loop out. Flame the lip of the tube again and put the cap back on. Incubate the slant at 37°C for 48 hrs and check the results. If the results seem negative, continue incubation for up to 96 hrs before reporting the results as negative.
-- Positive: Half or more of the medium is blackened (black to dark brown) in any time interval.
-- Negative: Less than half of the tube is blackened after 96 hrs.
Objective: To determine the ability of an organism to produce proteolytic-like enzymes (gelatinases) which break down gelatin. Gelatinase destroys (hydrolyzes) the gel and causes its collapse and liquefaction.
1. Obtain two solidified gelatin butts but keep them in the refrigerator until just prior to inoculation. Pick up a heavy inoculum from your pure culture and stab one of the butts to a depth of 2 inches. The other tube should not be inoculated and used as a control.
2. Incubate both the test and control tubes simultaneously at the optimal growth temperature for the organism for 48 hours to 14 days.
3. At the end of each 48-hour period, place both tubes (test bacterium and control) in a refrigerator for about 1 hour to determine whether digestion of gelatin (liquefaction) has occurred. Make the transfer from incubator to refrigerator without shaking the tubes. Check tubes daily up to 2 weeks unless liquefaction occurs sooner.
Gently tilt the tubes. The test is positive if the medium of the test organism is liquefied (gelatin breakdown) while that of the control has remained solid (lack of gelatin hydrolysis). The result of the test is negative if the medium of the test organism is as solid as that of the control.
-- Always run a control tube in parallel with organism being tested.
-- Gelatin is solid when incubated at 20°C or less and liquid at 30°C or greater. Gelatin changes from a gel (solid state) to a liquid at about 28°C. Therefore, if gelatin tubes are incubated at 30°C or greater, they must first be placed in a refrigerator for an hour and cooled before an interpretation of liquefaction is made.
-- Do not shake gelatin tubes while warm since growth and liquefaction of gelatin frequently occur only on the surface layer. If the gelatin is shaken and allowed to be mixed with the warm fluid of the medium, there is a possibility that a positive result may be overlooked, and thereby a false negative result may be obtained.
The first fluorescence-based flow cytometry device (ICP 11) was developed in 1968 by Wolfgang Göhde from the University of Münster, Germany and first commercialized in 1968/69 by German developer and manufacturer Partec through Phywe AG in Göttingen. At that time, absorption methods were still widely favored by other scientists over fluorescence methods. The original name of the flow cytometry technology was pulse cytophotometry (German: Impulszytophotometrie). Only 10 years later in 1978, at the Conference of the American Engineering Foundation in Pensacola, Florida, the name was changed to flow cytometry, a term that quickly became popular. Soon after, flow cytometry instruments were developed, including the Cytofluorograph (1971) from Bio/Physics Systems Inc. (later: Ortho Diagnostics), the PAS 8000 (1973) from Partec, the first FACS instrument from Becton Dickinson (1974), the ICP 22 (1975) from Partec/Phywe and the Epics from Coulter (1977/78).
Principle of flow cytometry
A beam of light (usually laser light) of a single wavelength is directed onto a hydrodynamically-focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam: one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and one or more fluorescent detectors).
Each suspended particle from 0.2 to 150 micrometers passing through the beam scatters the light in some way, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a longer wavelength than the light source. This combination of scattered and fluorescent light is picked up by the detectors, and, by analyzing fluctuations in brightness at each detector (one for each fluorescent emission peak), it is then possible to derive various types of information about the physical and chemical structure of each individual particle.
Har Gobind Khorana was born of Hindu parents in Raipur, a little village in Punjab, which is now part of eastern Pakistan. The correct date of his birth is not known; that shown in documents is January 9th, 1922. He is the youngest of a family of one daughter and four sons. His father was a «patwari», a village agricultural taxation clerk in the British Indian system of government. Although poor, his father was dedicated to educating his children and they were practically the only literate family in the village inhabited by about 100 people.
Har Gobind Khorana attended D.A.V. High School in Multan (now West Punjab); Ratan Lal, one of his teachers, influenced him greatly during that period. Later, he studied at the Punjab University in Lahore where he obtained an M. Sc. degree. Mahan Singh, a great teacher and accurate experimentalist, was his supervisor.
Khorana lived in India until 1945, when the award of a Government of India Fellowship made it possible for him to go to England and he studied for a Ph. D. degree at the University of Liverpool. Roger J. S. Beer supervised his research, and, in addition, looked after him diligently. It was the introduction of Khorana to Western civilization and culture.
Khorana spent a postdoctoral year (1948-1949) at the Eidgenössische Technische Hochschule in Zurich with Professor Vladimir Prelog. The association with Professor Prelog molded immeasurably his thought and philosophy towards science, work, and effort.
After a brief period in India in the fall of 1949, Khorana returned to England where he obtained a fellowship to work with Dr. (now Professor) G. W. Kenner and Professor (now Lord) A. R. Todd. He stayed in Cambridge from 1950 till 1952. Again, this stay proved to be of decisive value to Khorana. Interest in both proteins and nucleic acids took root at that time.
A job offer in 1952 from Dr. Gordon M. Shrum of British Columbia (now Chancellor of Simon Fraser University, British Columbia) took him to Vancouver. The British Columbia Research Council offered at that time very little by way of facilities, but there was «all the freedom in the world», to use Dr. Shrum's words, to do what the researcher liked to do. During the following years, with Dr. Shrum's inspiration and encouragement and frequent help and scientific counsel from Dr. Jack Campbell (now Head of the Department of Microbiology at the University of British Columbia), a group began to work in the field of biologically interesting phosphate esters and nucleic acids. Among the many devoted and loyal colleagues of this period, there should, in particular, be mention of Dr. Gordon M. Tener (now a Professor in the Biochemistry Department of the University of British Columbia), who contributed much to the spiritual and intellectual well-being of the group.
In 1960 Khorana moved to the Institute for Enzyme Research at the University of Wisconsin. He became a naturalized citizen of the United States. As of the fall of 1970 Khorana has been Alfred P. Sloan Professor of Biology and Chemistry at the Massachusetts Institute of Technology.
Har Gobind Khorana was married in 1952 to Esther Elizabeth Sibler, who is of Swiss origin. Esther brought a consistent sense of purpose into his life at a time when, after six years' absence from the country of his birth, Khorana felt out of place everywhere and at home nowhere. They have three children: Julia Elizabeth (born May 4th, 1953), Emily Anne (born October 18th, 1954), and Dave Roy (born July 26th, 1958).
A 52-year-old Malaysian man, a 24-year-old sub-Saharan woman, and a 28-year-old Madagascan woman (who was heterozygous for hemoglobin S) were admitted to North Hospital in Marseilles, France. Blood tests using an Advia2120i hematology analyzer (Siemens) showed no or mild anemia (109-150 g/L), normal or high mean corpuscular hemoglobin concentration (339-364 g/L), and borderline or slightly high red cell distribution width (15%-19.2%).
The red blood cell (RBC) volume and hemoglobin concentration cytogram clearly showed a typical distribution of comma-shaped RBCs, with an increased number of hyperchromic RBCs (panel A). Examination of the blood smear revealed anisocytosis and poikilocytosis, without spherocytes but with ovalocytes and macro-ovalocytes, some of them with more than 1 ridge (panel B; original magnification ×100, May-Grünwald Giemsa stain). The eosin-5′-maleimide binding test performed for each patient showed a reduced mean channel fluorescence between 26.2% and 30.9%, confirming an anomaly of the band-3 protein. A heterozygous 9-amino-acid deletion (residues 400 to 408) in band 3 (SLC4A1), which is the most common genetic abnormality in Southeast Asian ovalocytosis (SAO), was found in all 3 patients. Most cases of SAO are asymptomatic, so careful examination of a cytogram from the Advia2120i analyzer and close observation of the blood smear can help diagnose SAO.
Scientists at the University of York have harnessed the therapeutic effects of carbon monoxide-releasing molecules to develop a new antibiotic which could be used to treat the sexually transmitted infection gonorrhoea.
The infection, which is caused by the bacteria Neisseria gonorrhoeae, has developed a highly drug-resistant strain in recent years with new cases reported in the north of England and Japan.
There are concerns that gonorrhoea, which is the second most common sexually transmitted infection in England, is becoming untreatable.
Almost 35,000 cases were reported in England during 2014, with most cases affecting young men and women under the age of 25. The interdisciplinary team, from the University of York's Departments of Biology and Chemistry, targeted the "engine room" of the bacteria using carbon monoxide-releasing molecules (CO-RMs).
CO is produced naturally in the body, but there is increasing evidence that carbon monoxide enhances antibiotic action with huge potential for treating bacterial infections.
The scientists found that Neisseria gonorrhoeae is more sensitive to CO-based toxicity than other model bacterial pathogens, and may serve as a viable candidate for antimicrobial therapy using CO-RMs.
The CO molecule works by binding to the bacteria, preventing them from producing energy.
Scientists believe the breakthrough, published in the journal MedChemComm, could pave the way for new treatments.
Professor Ian Fairlamb, from the University's Department of Chemistry, said: "The carbon monoxide molecule targets the engine room, stopping the bacteria from respiring. Gonorrhoea only has one enzyme that needs inhibiting and then it can't respire oxygen and it dies.
"People will be well aware that CO is a toxic molecule but that is at high concentrations. Here we are using very low concentrations which we know the bacteria are sensitive to.
"We are looking at a molecule that can be released in a safe and controlled way to where it is needed."
The team say the next stage is to develop a drug, either in the form of a pill or cream, so that the fundamental research findings can be translated on to future clinical trials.
Professor Fairlamb added: "We think our study is an important breakthrough. It isn't the final drug yet but it is pretty close to it." "People might perceive gonorrhoea as a trivial bacterial infection, but the disease is becoming more dangerous and resistant to antibiotics."
The team worked with Professor James Moir from the University's Department of Biology. He added: "Antimicrobial resistance is a massive global problem which isn't going away. We need to use many different approaches, and the development of new drugs using bioinorganic chemistry is one crucial way we can tackle this problem, to control important bacterial pathogens before the current therapies stop working."
Chalita Suansane is a 21 year old Thailand native currently studying Microbiology at Mahasarakham University. Ever since Suansane was young, she was often curious and eager to learn new things, including a passion to explore living organisms that you cannot see by the eyes. On top of her studies, Suansane volunteers at ‘Baan Home Hug’ which is an orphanage that houses children who have inherited HIV from their parents, children who were abused, and children who have lost their family. If crowned, she would like to raise awareness and advocate for HIV/AIDS. Suansane would also like to let young women know that fulfilling your own passions, having self-respect, and being compassionate to others will make you confidently beautiful in your own way.
Description: As the study of embryology continues to be integrated with a range of disciplines, Before We Are Born remains the ideal solution for students who need to quickly learn the basics. Fully updated by the world’s foremost embryologists, this medical reference book provides concise guidance on human embryology at every stage of development, utilizing rich illustrations and photographs designed to further explain content.
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- File Name Before We Are Born: Essentials of Embryology and Birth Defects, 9th Ed.
- Edition 9th
- Year 2015
Keith L. Moore BA MSc PhD DSc FIAC FRSM FAAA, T. V. N. Persaud MD PhD DSc FRCPath (Lond.) FAAA, Mark G. Torchia MSc PhD
- ISBN-10 032331337X
- ISBN-13 978-0323313377
- Publisher Saunders; 9 edition (February 4, 2015)
- Size 23.5 MB
- File Format .pdf
- Password bioscience.pk
They are colored organic compounds used for staining microorganisms. Chemically,
Stains= Benzene ring+ Chromophore+ Auxochrome
According to nature of stain, it can be classified into:
1. Acidic Dyes: It is dye which has negative charge so they bind to positively charged cell structures like some proteins. Acidic dyes are not very often used in Microbiology lab.except to provide background staining like Capsule staining. Examples: Nigrosine, Picric acid, Eosin, Acid fuschin, India ink etc.
2. Basic Dyes: This dye have positive charge & bind to negatively charged molecules(nucleic acid, -COOH -OH). Since, surface of bacterial cells are negatively charged(due to Teichoic acid), basic dyes are most commonly used in bacteriology. Examples: Crystal Violet, Methylene Blue, Safranin , basic fuschin.
3. Neutral Dyes: They are usually formed from precipitation in which are produced when aqueous acidic & basic stains are combined. Neutral dyes stains nucleic acids, & cytoplasm. Eg; Eosinate of Methylene blue, Giesma stain.