WEEK 3: INTRODUCTION TO BACTERIA CULTURE AND TESTS Flashcards

Question

State how the following toxins produced by the Staphylococcus Auren's affect the host. Bacillus cereus enterotoxin Alpha hemolysin

Answer 1

1. Staphylococcal Enterotoxins These toxins cause food poisoning by affecting the gastrointestinal system. Bacillus cereus Enterotoxins (produced by Bacillus cereus): These toxins can cause symptoms of food poisoning when ingested. 2. Hemolysins: Alpha Hemolysin *Alpha hemolysin can lyse red blood cells and damage host tissues.

Answer 2

-Difference in growth requirements. -Colony morphology. -Metabolic reactions (& enzymes). -Virulence factors. -Resistance (or not) to antibiotics.

Answer 3

Devised by Danish bacteriologist, Hans Christian Gram, 1882 (published in 1884). To discriminate between pneumococci & Klebsiella pneumoniae in lung tissue.

Answer 4

Step 1. Label slide, place droplet of water & mix in colony. Heat fix colony (~30secs) Step 2. Addition of crystal violet (1 min, then rinse with H2O) In aqueous solution, crystal violet molecules dissociate into CV+ & Cl– ions which penetrate the bacterial cell wall. The CV+ ions interact with negatively charged components of the cell wall & membrane. Step 3. Addition of iodine (1 min, then rinse with H2O) Iodine is the mordant, the negatively charged iodine ions interact with CV+ ions & form CV-I complexes within the cytoplasm, cell membrane & cell wall. Enabling the cell wall to retain the crystal violet dye (“fixing the dye”) & preventing easy removal. All bacteria appear purple at this point. Step 4. Addition of decolorizer e.g., ethanol or acetone (~30secs, then rinse with H2O) The decolorizing solution (ethanol or acetone) interacts with lipids in the cell wall. *The lipids in the outer membrane of the Gram-ve bacterial are dissolved exposing the thin peptidoglycan layer (with less cross-linking) in the Gram-ve cell wall, so the primary dye more easily leaches into the solvent. Resulting in loss of most of the CV-I complexes & unstaining the cell wall. In contrast: *In Gram+ve bacteria, the peptidoglycan layer is much thicker (with higher cross-linkage), so less CV-I complexes are lost & as the decolorizing solution dehydrates the peptidoglycan, spaces within the cell wall ‘tighten’ & most CV-I complexes are retained NOTE: length of time of decolorization is critical, prolonged time results in removal of primary stain from the ‘Gram+ve’ cells too, leading to ‘false negatives’ Step 5. Addition of counterstain e.g., safranin or fuchsin (1 min, then rinse with H2O) When the positively charged safranin (counterstain) is added, it interacts with the free negatively charged components in Gram-ve cell wall & the membrane. *As there is no decolorizing step, the Gram-ve bacteria retain the pink/red color of the counterstain. *Whereas there is no space within the dehydrated cell wall of the Gram+ve bacteria due to the present CV-I complexes. So, the Gram+ve bacteria cannot retain the counterstain (safranin, so the Gram+ve bacteria retain the purple/violet color of the primary stain

Answer 5

To visualize microorganisms with atypical cell walls, that are not readily stained nor visualized by the Gram stain due to their unique cell wall composition, or absence of a cell wall e.g., Mycobacteria. Certain pathogens e.g., parasites, fungi, are viewed within clinical specimens i.e., & required special processing & histopathological staining of the clinical specimen in order to view them. Also, to enable visualization of different bacterial structures such as capsules, flagella, granules & spores, other staining methods are used.

Answer 6

Cell wall thick containing ~60% lipids Mycolic acids confer a waxy & hydrophobic consistency to cell surface. Making Mycobacterium impermeable to most stains including Gram staining (Nocardia & Rhod coccus)

Answer 7

So Acid fast stain is used: Carbolfuchsin (primary stain) Methylene blue (counter stain)

Answer 8

Step 1. Prepare a smear & heat fix Step 2. Carefully place slide over beaker of boiling water Step 3. Addition of Carbolfuchsin (5mins do not let it dry), carefully remove slide & with H2O) Heat & longer time facilitates penetration of the stain through lipid wall & into cytoplasm. All cells-stained red. Step 4: Decolorize with acid-alcohol. (1m, rinse with water) Acid fast cells with retain the primary stain while Non-acid fast cells will lose the primary stain. Step 5. Counterstain with methylene blue (30secs-1min, then rinse with H2O) Finally, viewing under the microscope with the oil immersion lens. Acid fast cells stain red with blue background, whereas non-acid fast cells stain blue.

Answer 9

1. Catalase test 2. Coagulase test 3. Bacitracin sensitivity test 4. DNase agar test Hemolysis of red blood cells

Answer 10

The catalase test is a microbiological test used to distinguish between bacteria that produce the enzyme catalase and those that do not. Catalase is an enzyme that helps break down hydrogen peroxide into water and oxygen. The test is particularly useful for differentiating between two major groups of bacteria: catalase-positive and catalase-negative.

Answer 11

1. Place a small colony of the bacterial culture on a clean microscope slide. 2. Add a small amount of 3% hydrogen peroxide to the bacterial colony on the slide. 3. Observe the reaction. *If the bacteria produce catalase, you will see the immediate production of bubbles (oxygen gas), indicating a positive result. Common Gram-positive bacteria that are catalase-positive include Staphylococcus species (except for Staphylococcus lugdunensis) and Micrococcus species. Catalase expressed by bacteria that need O2 for respiration (protects them from toxic oxygen metabolites Catalase-Negative Bacteria: Catalase-negative bacteria do not produce the catalase enzyme and do not produce bubbles when hydrogen peroxide is added. Catalase-ve bacteria may be anaerobes or facultative anaerobes & do not need O2 as a terminal electron acceptor These bacteria include many streptococci and enterococci.

Answer 12

In the context of clinical microbiology, hemolysis is often used to describe the breakdown of red blood cells in a culture medium due to the action of bacteria. There are three main types of hemolysis observed on blood agar plates: 1. Alpha Hemolysis: This is partial hemolysis, characterized by a greenish or brownish discoloration of the agar around bacterial colonies. It is typically caused by bacteria that produce hydrogen peroxide, which partially lyses the red blood cells. A greenish zone seen around colonies on BA. Due to bacterium expressing oxidization reaction of the iron in the hemoglobin within the RBC's. The RBC’s cell membrane remains intact, so α-hemolysis is not true lysis of RBC’s. 2. Beta Hemolysis: This is complete hemolysis, and it is characterized by a clear zone (no color) surrounding bacterial colonies on the blood agar plate. Beta-hemolytic bacteria produce enzymes, such as streptolysin, that completely lyse the red blood cells and break down hemoglobin. 3. Gamma Hemolysis: This is no hemolysis, and there is no change in the appearance of the agar around bacterial colonies. Gamma-hemolytic bacteria do not produce hemolysins and do not cause hemolysis.

Answer 13

Alpha () hemolysis Streptococcus pneumoniae viridians Streptococcus spp Beta () hemolysis Streptococcus pyogenes S. agalactiae (sometimes- Gamma () hemolysis -Enterococcus spp -Streptococcus Bovis

Answer 14

-GAS-Group Streptococcus pyogenes -GBS- Group B Streptococcus agalactiae

Answer 15

CAMP Test (Christie–Atkins–Munch-Peterson) CAMP test Presumptive identification of S. agalactiae (Group B Streptococcus-GBS). Only -hemolytic Streptococcus spp. with +ve CAMP test result Principle: S. agalactiae has a diffusible extracellular protein (CAMP factor) that diffuses into Blood agar. When it’s next to a -hemolytic Staphylococcus aureus strain, the CAMP factor acts synergistically with -hemolysin of S. aureus, enhancing the -hemolysis activity (NB. most effective with sheep or bovine BA media).

Answer 16

Bile (sodium deoxycholate) solubility test

Answer 17

Principle of the Bile Solubility Test: Streptococcus pneumoniae: This bacterium is known to be susceptible to bile salts. When exposed to sodium deoxycholate (bile salts), the cell wall of S. pneumoniae breaks down, causing the bacterial cells to lyse and disintegrate. As a result, the culture will become more translucent or "soluble." Other Alpha-Hemolytic Streptococci: Most other alpha-hemolytic streptococci are not as susceptible to bile salts. When exposed to sodium deoxycholate, their cell walls do not break down, and the culture remains intact. Procedure: 1. A bacterial colony from an isolated culture is suspended in a small amount of physiological saline (0.85% NaCl) to create a bacterial suspension. 2. A drop of sodium deoxycholate (bile salts) solution is added to the bacterial suspension. 3. Mix the suspension gently and observe the reaction. Interpretation: If the bacterial cells disintegrate and the culture becomes more translucent, this indicates a positive result for Streptococcus pneumoniae. If the culture remains intact, showing no significant change in opacity, this suggests a negative result, indicating a different alpha-hemolytic streptococcus species. The bile solubility test is a simple but effective method for identifying S. pneumoniae, which is important for clinical diagnosis and appropriate treatment decisions, particularly in cases of respiratory infections.

Answer 18

S. pneumoniae expresses an autolytic enzyme, an amidase triggering natural autolysis, in presence of bile salts. Whereas other α- hemolytic Streptococci do not lyse.

Answer 19

The lysis of DNA in the context of microbiology refers to the breakdown or degradation of DNA, typically used in a laboratory setting to identify and differentiate bacteria based on their ability to break down or hydrolyze DNA. The media used for this purpose is often called "DNA agar" or "DNA hydrolysis agar." Principle: In DNA agar, DNA from a variety of sources, such as DNA from fish sperm or other DNA-rich material, is incorporated into the agar medium. Some bacteria have the ability to produce deoxyribonucleases (DNases), enzymes that can hydrolyze or break down DNA. When these DNase-producing bacteria are streaked or inoculated onto DNA agar plates, they will grow and form colonies. As they grow, they release DNases that break down the DNA in the medium surrounding their colonies. Interpretation: Positive Result: Bacteria that produce DNases will show a clear zone of hydrolysis around their colonies. This clear zone is an indicator of DNA lysis. The larger the clear zone, the more efficient the organism is at hydrolyzing DNA. Negative Result: Bacteria that do not produce DNases will not create a clear zone around their colonies, and the DNA in the medium will remain intact. This test is useful in the identification and differentiation of bacteria, as some species, such as Staphylococcus aureus, are known to produce DNases. When inoculated on DNA agar, S. aureus will create a clear zone, while other bacteria without DNase activity will not. This test is particularly valuable in clinical microbiology for the identification of bacterial species.

Answer 20

Hydrochloric acid (HCl) is sometimes added to the DNA hydrolysis test (also known as DNase test) to adjust the pH of the agar medium. The addition of HCl serves a specific purpose: Controlled pH: -The activity of DNases (deoxyribonucleases) is pH-dependent. These enzymes are most active under acidic conditions. By adding HCl to the medium, the pH is lowered, creating an environment that is more conducive to the optimal activity of DNases produced by the bacteria being tested. Enhanced Hydrolysis: Lowering the pH in the medium allows for better and faster hydrolysis of the DNA incorporated into the agar. This results in more distinct and visible clear zones around the colonies of DNase-producing bacteria. In summary, the addition of HCl to the DNA hydrolysis test is done to create an acidic environment that optimizes the hydrolysis of DNA by DNases, facilitating the identification and differentiation of bacteria based on their DNase activity.

Answer 21

The tube coagulase test is a laboratory test used to determine the ability of certain bacteria, particularly Staphylococcus aureus, to produce an enzyme called coagulase. Coagulase is an enzyme that can cause the formation of blood clots, specifically by catalyzing the conversion of fibrinogen in the plasma into fibrin, which is a protein that is part of blood clots.

Answer 22

Here's how the tube coagulase test is typically performed: Inoculation: A bacterial colony, usually from an overnight culture, is inoculated into a tube containing plasma. The plasma used is often rabbit plasma. Incubation: The inoculated tube is then incubated at 37°C (body temperature) for several hours, usually around 4-24 hours. Observation: After incubation, the tube is examined for the presence of clot formation. If the plasma has clotted during the incubation period, it indicates that the bacterium produces coagulase and can cause the clotting of plasma. Interpretation: Positive Result: Clot formation in the tube during incubation is a positive result. This indicates the presence of coagulase and is often associated with Staphylococcus aureus. Staphylococcus aureus is the most well-known bacterium that produces coagulase. Negative Result: If there is no clot formation in the tube after incubation, it suggests a negative result, indicating that the tested bacterium does not produce coagulase. Many other staphylococci and bacteria do not produce coagulase. The tube coagulase test is valuable in clinical microbiology for the differentiation of Staphylococcus aureus from other staphylococci and is used as a part of the process of identifying and characterizing bacterial isolates in clinical laboratories.

Answer 23

Novobiocin resistance refers to the ability of bacteria to withstand the inhibitory effects of the antibiotic novobiocin. Novobiocin is an antibiotic that is used to treat bacterial infections, and it works by interfering with bacterial DNA replication and the function of DNA gyrase, an enzyme essential for DNA synthesis and replication. Staphylococcus saprophyticus, is a coagulase negative Staphylococci, which causes UTIs esp. in young adult female patients. S. saprophyticus is rarely resistant to most antibiotics but it’s innately resistant to novobiocin. 1. Zone of inhibition around novobiocin disk <16mm indicates bacterium is Resistant to the antibiotic. 2. Zone of inhibition around novobiocin disk >16mm=indicates bacterium is Susceptible to the antibiotic.

Answer 24

1. Modification of DNA Gyrase: Some bacteria develop mutations in their DNA gyrase enzyme, making it less susceptible to novobiocin's inhibitory effects. This allows the bacterium to continue its DNA replication unaffected by the antibiotic. 2. Efflux Pumps: Bacteria can develop efflux pumps that actively remove novobiocin from within the bacterial cell, preventing it from accumulating to effective levels. 3. Inactivation of Novobiocin: In some cases, bacteria may produce enzymes that chemically modify or inactivate novobiocin, rendering it ineffective.

Answer 25

The optochin test, also known as the optochin susceptibility test, is a laboratory test used to differentiate between alpha-hemolytic streptococci, which are a group of bacteria belonging to the Streptococcus genus. Specifically, the optochin test helps distinguish Streptococcus pneumoniae from other alpha-hemolytic streptococci, such as viridian's streptococci. Principle of the Optochin Test: The optochin test is based on the susceptibility of the tested bacteria to the antibiotic optochin (ethylhydrocupreine hydrochloride). Streptococcus pneumoniae is typically sensitive to optochin, while most other alpha-hemolytic streptococci are resistant to it. Procedure: 1. A blood agar plate is streaked with a pure culture of the alpha-hemolytic streptococcus to be tested. 2. Small optochin discs or optochin-containing disks are placed on the agar surface. 3. The plate is then incubated at 35-37°C in an atmosphere containing 5-10% carbon dioxide. Interpretation: *Zone of Inhibition: After incubation, the plate is examined for the presence of a clear zone of inhibition around the optochin disc. -The clear zone indicates that the bacteria are susceptible to optochin, and it is typically observed for Streptococcus pneumoniae. No Zone of Inhibition: If there is no clear zone around the optochin disc, it suggests resistance to optochin. This is typically seen in other alpha-hemolytic streptococci, such as various species of viridian's streptococci.

Answer 26

Optochin test

Answer 27

The bacitracin test, also known as the bacitracin susceptibility test, is a laboratory test used to differentiate between different species of beta-hemolytic streptococci, particularly Streptococcus pyogenes (Group A Streptococcus) and other beta-hemolytic streptococci. The test is based on the susceptibility of these bacteria to the antibiotic bacitracin. Principle of the Bacitracin Test: Streptococcus pyogenes (Group A Streptococcus) is typically sensitive to bacitracin, while most other beta-hemolytic streptococci are resistant to it. This test helps identify Group A Streptococcus among a mixed culture of beta-hemolytic streptococci.

Answer 28

Procedure: 1. A blood agar plate is streaked with a pure culture of the beta-hemolytic streptococcus to be tested. 2. A bacitracin disk or bacitracin-containing disc (typically 0.04 units of bacitracin) is placed on the agar surface. 3. The plate is then incubated at 35-37°C in an atmosphere containing 5-10% carbon dioxide. Interpretation: *Zone of Inhibition: After incubation, the plate is examined for the presence of a clear zone of inhibition around the bacitracin disc. The clear zone indicates that the bacteria are susceptible to bacitracin, which is typically seen in Streptococcus pyogenes (Group A Streptococcus). *No Zone of Inhibition: If there is no clear zone around the bacitracin disc, it suggests resistance to bacitracin. This is typically seen in other beta-hemolytic streptococci, such as Streptococcus agalactiae (Group B Streptococcus) and Streptococcus dysgalactiae.

Answer 29

Bacitracin test

Answer 30

“Litmus milk” is a milk-based media & litmus added as the pH indicator Allows for differentiation of bacteria due to varying metabolic activities of the milk components i.e., fermentation, reduction, clot/ curd & gas formation. Milk has lactose sugar, casein protein, vitamins & minerals, excellent for most microbial growth. Litmus is blue in alkaline conditions vs. Red/pink in acidic conditions.

Answer 31

1. Change in Color: -Purple/Pink: The litmus milk is usually purple or pink before inoculation. If the color remains unchanged or shifts toward pink after incubation, it indicates that the bacteria did not ferment lactose or produce acid. This result is often associated with organisms that lack lactase enzyme (lactose non-fermenters). -Light Blue: A shift to light blue or bluish green indicates the presence of alkaline products, suggesting that the organism has metabolized lactose and produced ammonia, which raises the pH of the medium. -Yellow/Clear: A shift to yellow or a clearing of the medium (reversion) indicates that the bacteria have fermented lactose and produced acid, which causes the medium to become acidic. 2. Clot Formation: Curds or Clots: The formation of curds or clots within the medium indicates that the bacteria have caused casein coagulation. This can be observed as solid or semisolid clots in the milk. Gas Production: 3. Gas Bubbles: Gas production in the form of bubbles or fissures in the medium may indicate the production of gas by certain bacteria during lactose fermentation. 4. Protein Digestion: Peptization: The digestion of milk proteins results in a clear, transparent liquid. This indicates the breakdown of milk proteins into peptides and amino acids. 5. Ejection and Sediment Formation: Whey Separation and Sediment: Whey separation with a sediment layer at the bottom of the tube may indicate proteolysis and peptonization of the milk, with the liquid whey separating from the solid sediment.

Answer 32

Enterobacteriaceae largest heterogenous Gram-positive rods/bacilli family. At times referred to as “enterics” as they are commensals of human colons.

Answer 33

Salmonella spp. Shigella spp. Escherichia coli Klebsiella spp Proteus spp. Yersinia pestis

Answer 34

Carbohydrates as the main energy source of many bacteria are metabolised by fermentation, producing: Acids i.e. lactic acid, formic acid, acetic acid, butyric acid, butyl alcohol, acetone, ethyl alcohol Gas i.e. CO2 & H2 Fermentation rxns are of great value in differentiating microorganisms i.e. Fermentation of the sugar with production of acid Fermentation with production of both acid + gas Sugars/ carbohydrates typically used in laboratory media incl. differential media: e.g., Lactose, Sucrose, Glucose

Answer 35

Specific carbohydrate e.g., glucose containing media is inoculated with a bacterium & observed for acid & /or gas formation. A pH indicator is typically added, in order to detect changes in pH.

Answer 36

Enterobacteriaceae spp. ferment glucose but fermentation of other sugars varies e.g. lactose. Lactose fermentation is used for differentiation of enteric bacteria. Enterobacteriaceae spp which are lactose fermenters are commonly ‘coliforms’ (‘specific term for a functional’ group of Enterobacteriaceae used as indicators of water quality) i.e. Escherichia, Klebsiella, Enterobacter, Hafnia & Citrobacter. Non-lactose fermenting are ‘non-coliform’ members i.e., Proteus, Morganella, Providencia, Salmonella, Shigella

Answer 37

Suspected Enterobacteriaceae spp. are initially grown on culture media: “enriched non-selective” media & “selective+differential” media. “Enriched non-selective” media e.g., Blood agar & Chocolate agar. Choc. agar is essentially blood agar but comprises lysed RBC’s, giving it a chocolate-brown color. Heating & lysis of RBC’s releases certain intracellular growth factors e.g., intracellular Nicotinamide adenine dinucleotide (NAD or V factor) & Hemin (factor X) needed by fastidious bacteria. E.g., Neisseria meningitidis & Hemophilus spp. are grown on Choc. Agar because they can’t grow in blood agar.

Answer 38

Selective and differential media are used in microbiology to isolate and differentiate specific groups of bacteria, such as enteric bacteria. Enteric bacteria are a group of Gram-negative, facultative anaerobic bacteria commonly found in the intestines of animals and humans. Some of the most well-known enteric bacteria include Escherichia coli (E. coli), Salmonella, and Shigella. Selective and differential media for enteric bacteria help in the selective growth of these organisms and differentiate them based on their metabolic characteristics.

Answer 39

Mackonkey agar *Selective - presence of crystal violet & bile salts inhibit growth of Gram-positive bacteria. *Differential - lactose fermenters vs. non-fermenters. Neutral red pH indicator – red/ pink at acidic pH & yellow at alkaline pH. 2. Eosin Methylene blue agar *Selective - Eosin Y & methylene blue inhibit growth of Gram-positive bacteria. -Differential – contains lactose & sucrose, vigorous fermenters produce acid. pH indicator - at low pH, Eosin Y & methylene blue combine to form a dark purple precipitate. Vigorous fermenters of lactose or sucrose produce acid sufficient to form a dark purple complex (with green metallic sheen. e.g., E. coli) Weak fermenters produce pink colonies. Colorless colonies: NON-fermenters of lactose &/or sucrose

Answer 40

*Oxidase test *Carbohydrates/ sugar fermentation tests i.e. glucose with Durham tubes *Methyl red/ Voges-Proskauer (MR/VP) *Citrate test *Motility test / Sulphur-Indole-Motility test *Triple Sugar Iron Agar / Kliger's Iron Agar *Urease test

Answer 41

The test is particularly useful in distinguishing between two major categories of bacteria: those that are oxidase-positive and those that are oxidase-negative. Used to identify microorganisms containing the enzyme cytochrome oxidase: The final stage of bacterial respiration involves the ‘electron transport chain.’ The final step in the electron transport chain may involve use of cytochrome oxidase, an enzyme that catalyzes oxidation of cytochrome c while reducing O2 to H2O For the oxidase test, reagent tetra-methyl-p-phenylenediamine dihydrochloride, is an artificial electron donor for cytochrome c. When the reagent is oxidized by cytochrome c, it changes from colorless to a dark blue or purple compound.

Answer 42

Oxidase-positive: Bacteria that contain cytochrome c oxidase are often aerobic or facultative anaerobes capable of using oxygen as the terminal electron acceptor in their electron transport chain. Many of these bacteria belong to the class Gammaproteobacteria, which includes various genera like Pseudomonas, Neisseria, and Campylobacter. Oxidase-negative: Bacteria that lack cytochrome c oxidase are typically fermenters or anaerobes that do not utilize oxygen as the terminal electron acceptor. Enterobacteriaceae, which includes genera like Escherichia, Salmonella, and Shigella, are examples of oxidase-negative bacteria.

Answer 43

Oxidase test can also be used to distinguish Gram-positive cocci: -Neisseria gonorrhoeae (oxidase +ve) -Staphylococcus spp. & Streptococcus spp. (oxidase -ve).

Answer 44

1. INDOLE TEST 2. METHYL RED-MR TEST 3. VOGES-PROSKAUER-VP 4. CITRATE USE TEST

Answer 45

Principle: to determine if bacterium can break down amino acid ‘tryptophan’ by action of tryptophanase into pyruvic acid, ammonia & indole. Media: Sulfide-indole-motility (SIM) medium or TSB After incubation & addition of Kovac’s rgt. POS=RED layer at top of tube NEG=no color change

Answer 46

The Methyl Red (MR) test is a biochemical test used in microbiology to determine the ability of certain bacteria to produce and maintain stable acids as a result of glucose fermentation. It is a part of a battery of tests used for the identification of bacteria and is commonly employed to differentiate between members of the Enterobacteriaceae family. Principle: Enterobacteriaceae spp. can convert glucose to pyruvic acid. Some further breakdown pyruvates by a ‘mixed acid pathway’, producing large amounts of stable mixed acids i.e., formic acid, acetic acid, lactic acid & succinic acid. Dropping the pH. *After adding MR rgt. pH indicator=RED (MR-POS) Some bacteria further decarboxylate the initial fermentation products & produce acetyl methyl carbinol (acetoin), which raises pH (≥pH 6.0). *After adding MR rgt. pH indicator =yellow (MR-NEG) Positive Result: If the culture medium turns red or remains red after the addition of the Methyl Red indicator, it indicates that the bacterium has produced stable acids from glucose fermentation. This is a positive MR test and suggests that the bacterium can maintain a low pH when fermenting glucose, producing significant amounts of mixed acids as a byproduct. Bacteria such as Escherichia coli often yield positive MR results. Negative Result: If the culture medium remains yellow (no color change) or turns yellow after the addition of the Methyl Red indicator, it indicates that the bacterium has not produced stable acids from glucose fermentation. This is a negative MR test, and it suggests that the bacterium does not produce significant mixed acids during glucose fermentation.

Answer 47

The Voges-Proskauer (VP) test is a biochemical test used in microbiology to detect the production of acetoin, a metabolic byproduct, and indirectly infer the presence of certain types of enteric bacteria, specifically those that can ferment glucose with a mixed acid fermentation pathway. MR & VP tests done simultaneously using same MR-VP broth. Bacteria fermenting sugars via the butanediol pathway produce acetoin as an intermediate which can be further reduced to 2,3-butanediol. After addition of KOH (VP rgt) it’s oxidized to diacetyl giving a pink/ed product=VP POS *Positive Result: The development of a red or pink color indicates a positive VP test. This color change is due to the presence of acetoin in the culture, which is produced as an intermediate in the fermentation of glucose. Bacteria that yield a positive VP test result have the ability to convert the end product of glucose fermentation, 2,3-butanediol, into acetoin in the presence of KOH and alpha-naphthol. This reaction is characteristic of organisms like Enterobacter and Serratia. *Negative Result: If there is no color change (the culture remains brown or copper), it indicates a negative VP test. This result suggests that the bacterium does not produce significant amounts of acetoin during glucose fermentation.

Answer 48

The Voges-Proskauer test, when used in combination with the Methyl Red test, helps differentiate between enteric bacteria based on their metabolic pathways. While MR identifies organisms that produce stable acids as a result of glucose fermentation, the VP test identifies those that produce acetoin. This information is valuable for the identification and classification of bacteria, particularly in the family Enterobacteriaceae.

Answer 49

The Citrate test is a biochemical test used in microbiology to determine the ability of bacteria to utilize citrate as their sole source of carbon and ammonium (NH4+) as their sole source of nitrogen. This test is typically used to differentiate between members of the Enterobacteriaceae family, such as Escherichia coli and Klebsiella pneumoniae. Bacteria that can grow on citrate agar, use it as a carbon source. Citrate is converted to pyruvate by citrate-permease & used for energy production. Ammonium phosphate is converted to ammonia & ammonium hydroxide, creating an alkaline pH. Bromothymol blue indicator turns from green to blue. Interpretation: *Positive Result: If the culture medium turns from its original green color to a deep blue color, it indicates a positive Citrate test. This color change suggests that the bacterium is capable of utilizing citrate as a carbon source and ammonium salts as a nitrogen source. The conversion of citrate to alkaline products (such as sodium carbonate) during citrate metabolism results in the increase of pH, leading to the color change to blue. *Negative Result: If there is no color change, and the agar remains green, it indicates a negative Citrate test. In this case, the bacterium cannot utilize citrate as a carbon source or ammonium salts as a nitrogen source.

Answer 50

SIM agar is semi-solid & contains sulphur, ferric ammonium citrate & tryptophan. It’s typically prepared in tubes referred to as stabs or deeps (not slanted). It’s used for three tests simultaneously: *Motility test: -Motile bacteria move in throughout the medium making it appear cloudy, stab line may appear ‘fuzzy’ or difficult to see. -Bacteria that are not motile: the stab line is clearly visible, no cloudiness. *Indole test: after addition of Kovac’s rgt, color change to deep pink at the meniscus = POS *Hydrogen sulfide production: a dark grey/ black ppt formed = POS Bacteria are inoculated by ‘stabbing’ the medium.

Answer 51

Triple Sugar Iron (TSI) agar is a differential and selective microbiological medium commonly used for the identification of enteric bacteria, particularly those belonging to the family Enterobacteriaceae. This versatile medium provides information about the fermentation of sugars, gas production, and sulfur reduction by bacteria. Here's how the Triple Sugar Iron agar test is typically performed: Procedure: Stab the TSI agar with a straight inoculation needle or wire to create a deep stab down the center of the medium. Streak the surface of the agar slant using an inoculating loop, creating a "streak" pattern. Incubate the TSI agar tube at the appropriate temperature, typically around 35-37°C, for 18-24 hours. Interpretation: Fermentation of Sugars: Yellow Slant (upper portion): If the slant portion of the TSI agar turns yellow, it indicates the fermentation of glucose. Most enteric bacteria are capable of fermenting glucose, so this part of the medium often turns yellow. Yellow Butt (lower portion): If the butt portion of the TSI agar turns yellow, it indicates the fermentation of lactose or sucrose. The color change in the butt is typically due to the accumulation of acid produced during lactose or sucrose fermentation. Gas Production: If there is a crack or lifting of the agar within the medium (accompanied by the presence of gas bubbles), it indicates gas production. Gas is produced as a byproduct of sugar fermentation. Sulfur Reduction: Black Precipitate: A black color in the medium, particularly in the butt of the tube, indicates sulfur reduction. The reduction of sulfur in the medium produces hydrogen sulfide (H2S), which reacts with iron salts in the medium to form a black precipitate. The TSI agar test is an essential tool in clinical microbiology, particularly in the identification of pathogens and in the surveillance of food safety and hygiene, where the presence of specific enteric bacteria can indicate contamination or spoilage.

Answer 52

Used to differentiate Enterobacteriaceae species, based on: Carbohydrate fermentation TSI contains three sugars: 1% lactose, 1% sucrose, 0.1% glucose Hydrogen sulfide production TSI also contains sodium thiosulfate & ferrous sulfate or ferrous ammonium sulfate Addition of pH indicator shows differentiation of bacteria based on ability (or lack of) to: Metabolize lactose &/ or sucrose &/ or glucose. Production of acid &/ or gas

Answer 53

TSI: Tripple Sugar Iron test

Answer 54

Differentiate Enterobacteriaceae species by determining the ability (or lack of) to hydrolyze urea producing ammonia & CO2 , by the activity of urease. Formation of ammonia alkalinizes the medium & changes the pH causing the pH indicator to change color from light orange at to deep pink.

Answer 55

The Kirby-Bauer test, also known as the disc diffusion method, is a widely used technique in clinical microbiology to determine the susceptibility of bacteria to specific antimicrobial agents. This test helps clinicians and microbiologists choose the most effective antibiotic for treating bacterial infections. It is a qualitative test that provides information about the sensitivity or resistance of a bacterial isolate to a particular antibiotic.

Answer 56

Procedure: A pure culture of the bacterium being tested is spread evenly on the surface of a Mueller-Hinton agar plate using a sterile swab. The density of the bacterial lawn should be adjusted to meet the guidelines set by the Clinical and Laboratory Standards Institute (CLSI) or other relevant authorities. Antimicrobial discs containing known concentrations of specific antibiotics are placed on the surface of the agar. Each disc contains a different antibiotic. The choice of antibiotics on the discs depends on the clinical suspicion and the organism being tested. The agar plate is then incubated at the recommended temperature and for a specific duration, typically 18-24 hours at 35-37°C. After incubation, the plate is examined for zones of inhibition (clear areas) around each antibiotic disc. The diameter of these zones is measured and compared to interpretive criteria provided by the CLSI or other relevant authorities.

Answer 57

Interpretation: The size of the zones of inhibition is used to categorize the bacteria's susceptibility or resistance to the tested antibiotics. The zones are typically categorized as follows: *Susceptible (S): If the diameter of the zone of inhibition falls within the range specified by the interpretive criteria, the bacterium is considered susceptible to the antibiotic. The antibiotic is likely to be effective in treating an infection caused by this organism. Intermediate (I): An intermediate result suggests that the antibiotic may be effective at higher doses or for specific types of infections. It is considered a borderline result and requires careful clinical consideration. Resistant (R): If the zone of inhibition is smaller than the range specified by the interpretive criteria, the bacterium is considered resistant to the antibiotic. This antibiotic is unlikely to be effective in treating infections caused by this organism.

Answer 58

1. Comparison of zone sizes to standardized thresholds only provides info. on whether the bacterium is susceptible or resistant to tested antibiotics. *Can’t distinguish between bacteriostatic & bactericidal activities of respective antibiotics. 2. Zone sizes are not an interpretation of the potency of the tested antibiotics. *So, the KB test can’t be used for comparing the potencies of different antibiotics & guide the best therapy. WHAT IS POTENCY? *In pharmacology, "potency" refers to the strength or effectiveness of a drug or therapeutic agent in producing a specific biological or pharmacological response. It measures the amount or concentration of a drug required to achieve a desired effect, typically at a specific level or extent. 3. Several factors affect the zone of inhibition size incl.: *Solubility of the antibiotic *Rate of diffusion through agar *Thickness of the agar *Conc. of the antibiotic in the disk

Answer 59

Antimicrobial dilution test: Several tubes with increasing concentration of antibiotic are inoculated with the bacterium. After incubation: *Cloudiness indicates growth of the bacterium at that conc. of the antibiotic. *Clear medium indicates, bacterium growth inhibited at that conc. of antibiotic.

Answer 60

Minimal Inhibitory Concentration (MIC): - Lowest conc. of an antibiotic that inhibits visible bacterial growth. Minimal Bactericidal Concentration (MBC) - Lowest conc. of the antibiotic that kills ≥99.9% of the starting inoculum Determining MIC & MBC helps to identify the most optimal antibiotic for a particular bacterium.

Answer 61

The lowest dilution of the antimicrobial that inhibits cloudiness (turbidity) is the ‘Minimal Inhibitory Concentration’ (MIC). Then The broth from tubes without turbidity can be inoculated onto culture media plates lacking the antimicrobial drug. The lowest conc. that shows killing of ≥99.9% of the starting inoculum is the ‘Minimal Bactericidal Concentration’ (MBC).

Answer 62

1. A combination of the Kirby-Bauer disk diffusion test & dilution methods 2. Strip containing a gradient conc. of an antibiotic E-tests strips are placed on the surface of a media plate inoculated with the bacteria. After incubation: confluent growth of the microorganism is visible. The MIC is read where inhibition zone intersects at the side of the strip. 3. E-test (previously known as Epsilometer test). 4. As determines whether or not the tested microorganism is susceptible or resistant to a specific antimicrobial & also determines MICs Commonly used to help guide clinicians on optimal treatment for patients. As it shows conc. of antimicrobial inhibits the growth of the pathogen causing the infection.

Answer 63

The MBC cannot be determined from the E-test.