BIO203A Lab Final Exam Study Guide PDF

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LAB FINALS STAINING 1.DESCRIBE THE PRINCIPLES BEHIND THE GRAM STAIN 2.DESCRIBE THE STEPS, REAGENTS AND PROCEDURES REQUIRED TO A GRAM STAIN

Principle of Gram staining The structure of the organism’s cell wall determines whether the organism is gram positive or negative. When stained with a primary stain and fixed by a mordant, some bacteria are able to retain the primary stain by resisting decolorization while others get decolorized by a decolorizer. Those bacteria which retain the primary stain are called Gram positive and those bacteria which gets decolorized and then get counterstained are called Gram negative.

Gram Staining : Principle, Procedure, Interpretation and Animation

The Gram staining technique is the most important and widely used microbiological differential staining technique. It was developed by Dr. Christian Gram in 1884, and categorizes bacteria according to their Gram character (Gram positive or Gram negative). In addition this stain also allows determination of cell morphology, size, and arrangement. It is typically the first differential test run on a specimen brought into the laboratory for identification. In some cases, a rapid, presumptive identification of the organism or elimination of a particular organism is possible

Crystal violet (CV) dissociates into CV+ and Cl– ions in aqueous solutions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple.

Iodine (I), used as mordant interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. Since Gram negative organism have thin peptidoglycan layer(1-2 layers) and have

additional lipopolysaccharide layer which gets dissolved due to the addition of alcohol, so gram negative organism fails to retain the complex and gets decolorized as the complex is washed away. In contrast, a Gram-positive cell becomes dehydrated from an ethanol treatment. This closes the pores in the cell wall and prevents the stain from exiting the cell. The large CV–I complexes become trapped within the Gram-positive cell also due to the thick and multilayered (40 layers) nature of its peptidoglycan

Reagents Used in Gram Staining    

Crystal Violet, the primary stain Iodine, the mordant A decolorizer made of acetone and alcohol (95%) Safranin, the counterstain

Gram Staining : Principle, Procedure, Interpretation and Animation by Editorial Team on January 8, 2020 in Bacteriology, Microbiology

The Gram staining technique is the most important and widely used microbiological differential staining technique. It was developed by Dr. Christian Gram in 1884, and categorizes bacteria according to their Gram character (Gram positive or Gram negative). In addition this stain also allows determination of cell morphology, size, and arrangement. It is typically the first differential test run on a specimen brought into the laboratory for identification.

After decolorization, the Gram-positive cell remains purple and the Gram-negative cell loses its purple color. Counterstain, which is usually positively-charged safranin or basic fuchsin, is applied last to give decolorized Gram-negative bacteria a pink or red color Procedure of Gram staining Smear preparation : 1. 2. 3.

Take a grease free dry slide. Sterilize the inoculating loop on a flame of a Bunsen burner. Transfer a loopful of culture (or the specimen) by sterile loop and make a smear at the center. Smear should not be very thin or very thick. 4. Allow the smear to dry in the air. 5. Fix the dry smear by passing the slide 3-4 times through the flame quickly with the smear side facing up.

Gram Staining :

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Place the slides on the staining rods. Cover the smear with crystal violet stain and leave for 1 minute. Wash carefully under running tap water. Flood the smear with Gram’s iodine solution and leave for 1 minute. Drain off the iodine Wash the slide for the again in a gentle stream of tap water. Flood the slide with the decolorizing agent then wait for 20-30 seconds. This can also be done by adding a drop by drop to the slide until the decolorizing agent running from the slides runs clear. Gently wash the slide under running tap water and drain completely. Counterstain with safranin for and and wait for about 30 seconds to 1 minute. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then blot dry with absorbent paper. Observe under microscope.

Gram Staining : Principle, Procedure, Interpretation and Animation by Editorial Team on January 8, 2020 in Bacteriology, Microbiology

The Gram staining technique is the most important and widely used microbiological differential staining technique. It was developed by Dr. Christian Gram in 1884, and categorizes bacteria according to their Gram character (Gram positive or Gram negative). In addition this stain also allows determination of cell morphology, size, and arrangement. It is typically the first differential test run on a specimen brought into the laboratory for identification. In some cases, a rapid, presumptive identification of the organism or elimination of a particular organism is possible. 3. BE ABLE TO TROUBLE SHOOT AND INTERPRET FINDINGS OBTAINED FROM A GRAM STAINED SPECIMEN Interpretation of Gram staining

The staining results of gram stain are as follows :    

Gram Positive : Dark purple Gram Negative : Pale to dark red Yeasts : Dark purple Epithelial cells : Pale red

Examples of Gram Positive Organisms Bacillus, Nocardia, Clostridium, Propionibacterium, Actinomyces, Enterococcus, Cornyebacterium, Listria, Lactobacillus, Gardnerella, Mycoplasma, Staphylococcus, Streptomyces, Streptococcus etc Examples of Gram Negative Organisms Escherichia, Helicobacter, Hemophilus, Neisseria, Klebsiella, Enterobacter, Chlamydia, Vibrio, Pseudomonas, Salmonella, Shigella ISOLATION AND CULTURE / CHARACTERISTICS OF MICROBES 4. DESCRIBE THE ENZYMATIC AND GROWTH CHARACTERISTICS OF AEROBIC AND ANAEROBIC MICROBES IN AGAR Aerobic Bacteria These are the species of bacteria which require oxygen for their basic survival, growth, and the process of reproduction. It is very easy to isolate these bacteria by culturing a mass of bacterial strains in some liquid medium. As they require oxygen for survival, they tend to come to the surface in a bid to derive maximum oxygen available. Examples of aerobic bacteria. » Bacillus » Nocardia Anaerobic Bacteria Also referred to as anaerobes, these are the species of bacteria which don’t require oxygen for growth. There are different types of anaerobic species, including the aerotolerant anaerobes, which can survive in the presence of oxygen, and obligate anaerobes, which can’t survive in the presence of oxygen.

Examples of anaerobic bacteria. » Escherichia coli » Bacteroides 5.DISTINGUISH BETWEEN WHICH TYPES OF MEDIA ARE SELECTIVE, DIFFERENTIAL OR BOTH OR NONE SELECTIVE AND DIFFERENTIAL MEDIA Selective and differential media are used to isolate or identify particular organisms. Selective media allow certain types of organisms to grow, and inhibit the growth of other organisms. The selectivity is accomplished in several ways. For example, organisms that can utilize a given sugar are easily screened by making that sugar the only carbon source in the medium Differential media are used to differentiate closely related organisms or groups of organisms. Owing to the presence of certain dyes or chemicals in the media, the organisms will produce characteristic changes or growth patterns that are used for identification or differentiation 6. LIST OR PREDICT WHICH MICROBES GROW ON DIFFERENT TYPES OF SELECTIVE AND DIFFERENTIAL MEDIA (MICROBES REFERENCED ON POWERPOINT) 7. DESCRIBE THE COMPONENTS OF THESE MEDIA MANNITOL SALT AGAR (MSA) Mannitol salt agar is a selective medium used for the isolation of pathogenic staphylococci. The medium contains mannitol, a phenol red indicator, and 7.5% sodium chloride. The high salt concentration inhibits the growth of most bacteria other than staphylococci. On MSA, pathogenic Staphylococcus aureus produces small colonies surrounded by yellow zones. The reason for this change in color is that S. aureus ferments the mannitol, producing an acid, which, in turn, changes the indicator from red to yellow. EOSIN METHYLENE BLUE AGAR (EMB agar) Eosin methylene blue agar is a differential medium used for the detection and isolation of Gram-negative intestinal pathogens. A combination of eosin and methylene blue is used as an indicator and allows differentiation between organisms that ferment lactose and those that do not. Saccharose is also included in the medium because certain members of the Enterobacteria or coliform group ferment saccharose more readily than they ferment lactose. In addition, methylene blue acts as an inhibitor to Gram-positive organisms. MacCONKEY'S AGAR MacConkey's agar is a differential plating medium used in the detection and isolation of all types of dysentery, typhoid and paratyphoid organisms. It is generally used for differentiating strains of Salmonella typhosa from members of the coliform group; however, the medium supports the growth of all Salmonella and Shigella strains and gives good differentiation between these enteric pathogens and the coliform group. When grown on MacConkeyís medium, colonies of coliform bacteria are brick-red in color and are surrounded by a zone of precipitated bile. The acid end-products act on bile salts, and neutral red is absorbed by the precipitated salts. Dysentery, typhoid and paratyphoid bacilli do not ferment lactose but give an alkaline reaction when grown on the medium. Colonies of these organisms are noncolored and transparent. The growth of Gram- positive organisms is inhibited because of the crystal violet and bile salts in the medium.

8. BE ABLE TO INTERPRET RESULTS FROM SUCH MEDIA (WHAT TYPE OF MICROBES GROW IN SOME CASES AND IN OTHERS YOU NEED TO KNOW THE PRECISE SPECIES THAT GROW OUT AND OTHER CASES WHICH MICROBES DIE OFF The number of available media to grow bacteria is considerable. Some media are considered general all-purpose media and support growth of a large variety of organisms. A prime example of an all-purpose medium is tryptic soy broth (TSB). Specialized media are used in the identification of bacteria and are supplemented with dyes, pH indicators, or antibiotics. One type,

enriched media contains growth factors, vitamins, and other essential nutrients to promote the growth of fastidious organisms, organisms that cannot make certain nutrients and require them to be added to the medium. When the complete chemical composition of a medium is known, it is called a chemically defined medium. For example, in EZ medium, all individual chemical components are identified and the exact amounts of each is known.

In complex media, which contain extracts and digests of yeasts, meat, or plants, the precise chemical composition of the medium is not known. Amounts of individual components are undetermined and variable. Nutrient broth, tryptic soy broth, and brain heart infusion are all examples of complex media.

Media that inhibit the growth of unwanted microorganisms and support the growth of the organism of interest by supplying nutrients and reducing competition are called selective media. An example of a selective medium is MacConkey agar. It contains bile salts and crystal violet, which interfere with the growth of many gram-positive bacteria and favor the growth of gram-negative bacteria, particularly the Enterobacteriaceae. These species are commonly named enterics, reside in the intestine, and are adapted to the presence of bile salts.

The enrichment cultures foster the preferential growth of a desired microorganism that represents a fraction of the organisms present in an inoculum. For example, if we want to isolate bacteria that break down crude oil, hydrocarbon clastic bacteria, sequential subculturing in a medium that supplies carbon only in the form of crude oil will enrich the cultures with oil-eating bacteria.

The differential media make it easy to distinguish colonies of different bacteria by a change in the color of the colonies or the color of the medium. Color changes are the result of end products created by interaction of bacterial enzymes with differential substrates in the medium or, in the case of hemolytic reactions, the lysis of red blood cells in the medium. In Figure 9.32, the differential fermentation of lactose can be observed on MacConkey agar. The lactose fermenters produce acid, which turns the medium and the colonies of strong fermenters hot pink. The medium is supplemented with the pH indicator neutral red, which turns to hot pink at low pH. Selective and differential media can be combined and play an important role in the identification of bacteria by biochemical methods.

Isolation and Culture and Characterization of Microbes 9. Describe the principles behind the catalase, coagulase and bile salts. The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water. The presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen peroxide, and the rapid elaboration of oxygen bubbles occurs. The lack of catalase is evident by a lack of or weak bubble production. The culture should not be more than 24 hours old.

Bacteria thereby protect themselves from the lethal effect of Hydrogen peroxide which is accumulated as an end product of aerobic carbohydrate metabolism. Coagulases are enzymes that clot blood plasma by a mechanism that is similar to normal clotting. The coagulase test identifies whether an organism produces this exoenzyme. This enzyme clots the plasma component of blood. The only significant disease-causing bacteria of humans that produce coagulase enzyme are Staphylococcus aureus. Thus, this enzyme is a good indicator of the pathogenic potential of S. aureus. In human host, the action of coagulase enzyme produces clotting of the plasma by converting fibrinogen to fibrin in the immediate vicinity of the bacterium as a means of protection by itself. The fibrin meshwork that is formed by this conversion surrounds the bacterial cells or infected tissues, protecting the organism from non-specific host resistance mechanisms such as phagocytosis and the anti-staphylococcal activity of normal serum. This enables the bacterium to persist in the presence of a host immune response, which can lead to the establishment of infection. Thus, coagulase is described as a virulence factor (disease- causing factor) of Staphylococcus aureus. Citrate and EDTA are usually added to act as anticoagulants and prevent false-positive results.

Bile salts are the major organic solutes in bile and normally function to emulsify dietary fats and facilitate their intestinal absorption. Bile is the major route for elimination of cholesterol. Bile salts are composed of the salts of four different kinds of free bile acids (cholic, deoxycholic, chenodeoxycholic, and lithocholic acids); each of these acids may in turn combine with glycine or taurine to form more complex acids and salts. Bile salts and acids can be synthesized from cholesterol or extracted from the bloodstream by the liver. They pass from the liver into the small intestine, where they act as detergents to emulsify fat and reduce the surface tension on fat droplets to prepare them for the action of pancreatic and intestinal fat-splitting enzymes. The salts are large, negatively charged ions that are not readily absorbed by the upper region of the small intestine; consequently, they remain in the small intestine until most of the fat is digested. In the lower small intestine, the salts and acids are absorbed and passed back into the bloodstream until they are once again extracted by the liver; this cycle, from the liver to the small intestine and blood and then back to the liver, is called enterohepatic circulation. Some salts and acids are lost during this process; these are replaced in the liver by continual synthesis from cholesterol. The rate of synthesis is directly related to the amount of acids and salts lost. Bile salts do not normally reach the colon; when they do, however, they may inhibit the absorption of water and sodium, causing a watery diarrhea.

10. Describe how these tests work, how to do them and when to perform such tests and what they tell you (know what microbes are identified upon being subjected to these tests).

Purpose or Uses of Catalase Test The morphologically similar Enterococcusor Streptococcus (catalase negative) and Staphylococcus (catalase positive) can be differentiated using the catalase test.  Also valuable in differentiating aerobic and obligate anaerobic bacteria.  Semiquantitative catalase test is used for the identification of Mycobacterium tuberculosis.  It is used to differentiate aerotolerant strains of Clostridium, which are catalase negative, from Bacillus species, which are positive.  Catalase test can be used as an aid to the identification of Enterobacteriaceae. Procedure of Catalase Test 

Tube Method 1. Pour 1-2 ml of hydrogen peroxide solution into a test tube. 2. Using a sterile wooden stick or a glass rod, take several colonies of the 18 to 24 hours test organism and immerse in the hydrogen peroxide solution. 3. Observe for immediate bubbling. Slide Method 1. Use a loop or sterile wooden stick to transfer a small amount of colony growth in the surface of a clean, dry glass slide. 2. Place a drop of 3% H2O2 in the glass slide. 3. Observe for the evolution of oxygen bubbles.

Procedure and Types of Coagulase Test Slide Test (to detect bound coagulase) 1. Place a drop of physiological saline on each end of a slide, or on two separate slides. 2. With the loop, straight wire or wodden stick, emulsify a portion of the isolated colony in each drops to make two thick suspensions. 3. Add a drop of human or rabbit plasma to one of the suspensions, and mix gently. 4. Look for clumping of the organisms within 10 seconds. 5. No plasma is added to the second suspension to differentiate any granular appearance of the organism from true coagulase clumping.

Tube Test (to detect free coagulase) 1. Dilute the plasma 1 in 10 in physiological saline ( mix 0.2 ml of plasma with 1.8 ml of saline). 2. Take 3 small test tubes and label as T (Test), P (Positive Control) and N (Negative Control). Test is 18-24 hour broth culture, Positive control is 18-24 hr S. aureus broth culture and Negative control is sterile broth. 3. Pipette 0.5 ml of the diluted plasma into each tube. 4. Add 5 drops (0.1 ml) of the Test organisms to the tube labelled “T”, 5 drops of S. aureus culture to the tube labelled “P” and 5 drops of sterile broth to the tube labelled “N”. 5. After mixing, incubate the three tubes at 35-37 Degree Celsius. 6. Examine for clotting after 1 hours. If no clotting has occurred, examine at 30 minutes intervals for up to 6 hours.

Bile is generated by the liver and is held by the gall bladder. Whenever food is ingested, bile is released into the duodenum. Bile salts formation is initiated with the disintegration of red blood cells. Rupturing of the damaged and old red blood cells is brought about as they pass through the liver or spleen.

Iron is removed and hemoglobin is disintegrated by the macrophages from the heme element. This portion of the heme which is free from iron is transformed into a green pigment known as biliverdin and subsequently into a yellow-orange pigment known as bilirubin. The bilirubin in the liver is extracted as a bile pigment that moves to the small intestine and further into the large intestine. 1. Smith’s Test Material Required 

Smith’s reagent

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