WEEK 3: INTRODUCTION TO BACTERIA CULTURE AND TESTS

Question 1

Define Clinical or medical microbiology.

Answer 1

Systematic investigation & identification of microorganisms that cause disease / infections (pathogens).

Question 2

Microorganisms can cause various infections affecting different anatomical sites. Name the disease and bacteria described below.

1. Causes sore throat, tonsillitis (gray patches in mouth)
2. Infection of endocardium &/or heart valves
3. Spores enter skin, germinate & cause lesions.
4. Causes foodborne illness esp. with immunocompromised.

Answer 2

1. Diphtheria: Corynebacterium diphtheria causes sore throat, tonsillitis (gray patches in mouth)
2. Endocarditis: Staphylococcus spp. Enterococcus spp. infection of endocardium &/or heart valves
3. Cutaneous anthrax: Bacillus anthracis spores enter skin, germinate & cause lesions.
4. Listeriosis: Listeria monocytogenes causes foodborne illness esp. with immunocompromised.

Question 3

Specimens collected from patients in clinics &/or hospitals are representative of the patient in the lab.

Give examples of what these specimens could be.

Answer 3

e.g., blood, urine, sputum, stool, aspirates (e.g., lung, respiratory, joint), swabs, biopsies, cerebral spinal fluid

Question 4

State the 3 diagnostic techniques done in a microbiology lab.

Answer 4

*Microscopy (with or without staining)
*Culture
*Tests:
Biochemical; Serological; Molecular, Antibiotic Susceptibility

Question 5

Failure to identify pathogens is NOT definitive of no infection. What could that indicate?

Answer 5

Failure to identify pathogens is NOT definitive of no infection. Rather it may indicate:

*Scant presence of pathogen (due to stage of disease/infection)
*Compromised viability during times of collection & delivery to lab
*Inadequate method of testing

Question 6

Bacteria traditionally categorized as Gram positive, or Gram negative based on the color they retain after completion of the Gram’s stain.

What is the color staining for Gram-positive bacteria?

What is the color staining for Gram-negative bacteria?

Answer 6

Gram+ve bacteria retain the purple/dark blue color from primary dye ‘Crystal violet.’

Gram-ve bacteria retain the red/pink color from the counterstain dye ‘Safranin.

Question 7

With the Gram’s stain the shape of the bacteria is also evident & generally bacteria are classified as:

Answer 7

*Rods (bacilli)
*Spheres (cocci)
Cocci bacilli, spirals, helixes, filaments etc.

Question 8

Describe Enterobacterales.

Answer 8

*Enterobacterales is an order of Gram-negative *Facultatively anaerobic
*Non-spore-forming
*Rod-shaped microorganisms.

Question 9

Enterobacterales has 7 families, of which the family Enterobacteriaceae (Gram–ve bacteria family residing in large intestine) contains species of clinical relevance.

State the leading 5 pathogens from Enterobacteriaceae and the diseases they cause.

Answer 9

Escherichia coli: urinary tract infections (UTIs), gastroenteritis

Klebsiella spp.: lung, meningitis, UTIs & bacteraemia

Shigella spp.: gastroenteritis

Salmonella spp.: gastroenteritis, typhoid fever

Proteus spp.: UTIs, gastroenteritis

Question 10

State some Gram-ve bacterial infections and the diseases they cause.

Answer 10

Pseudomonas aeruginosa: cystis fibrosis & UTIs

Acinetobacter baumannii: soft tissue infections

Mycobacterium tuberculosis: TB

Neisseria gonorrhoeae: STDs

Question 11

Give examples of Gram-positive bacterial infections and the diseases that they cause.

Answer 11

1. Otitis Media (middle ear infection) Streptococcus pneumoniae
2. Diphtheria: Corynebacterium diphtheria causes sore throat, tonsillitis (gray patches in mouth)
3. Listeriosis: Listeria monocytogenes causes foodborne illness esp.
4. Pneumonia: Streptococcus pneumoniae lung infection
5. Endocarditis: Staphylococcus spp. Enterococcus spp. infection of endocardium &/or heart valves
6. Cutaneous anthrax: Bacillus anthracis spores enter skin & cause lesions

Question 12

State the 3 Gram+ve cocci which generally a leading cause of many human infections.

Answer 12

Staphylococci
Streptococci
Enterococci

Question 13

State the 4 examples of Gram-positive rods/bacilli species.

Answer 13

Listeria species
BacilIus species
Corynebacterium species
Propionibacterium species

Question 14

State the 3 streptococcus species infections.

Answer 14

S. pneumoniae
S. pyogenes
S. agalactiae
S. epidermidis

Question 15

What disease does S. pneumoniae AND
S. pyogenes cause?

Answer 15

S. pyogenes: pharyngitis (A)
S. pneumoniae: pneumonia (B)

Question 16

State the 3 diseases caused by S. Auren’s.

Answer 16

S. aureus:
osteomyelitis
surgical site infections
folliculitis

Question 17

State the 2 Enterococcus species.

Answer 17

E. faecalis
E. faecium

Question 18

State the disease caused by E. faecalis.

Answer 18

E. faecalis: urinary tract infections

Question 19

Gram+ve bacteria are increasingly resistant to antibiotics.

Give 2 examples of Gram-positive antibiotic resistant bacteria.

Answer 19

*Methicillin-resistant Staphylococcus aureus (MRSA)
*Vancomycin-resistant Enterococci

Question 20

What is the function of virulence factors?

Give an example.

Answer 20

Virulence factors: augment ability to cause infections (pathogenicity); magnitude & duration of infections.

*Cell wall carbohydrates & proteins mediate bacterial attachment (& tissue tropism).
*Factors that enable evasion & survival in the immune system.
*Toxins.
*Hydrolytic enzymes

Question 21

State the 2 virulence factors that help in invasion by bacteria.

Describe how they achieve that.

Answer 21

*Streptokinase:
HOW?

1. Fibrinolytic Activity:
   Streptokinase is an enzyme that has fibrinolytic activity, which means it can break down fibrin, a protein involved in blood clot formation.

By breaking down blood clots, streptokinase helps bacteria to dissolve the fibrin mesh in the blood, promoting the spread of the bacteria within the bloodstream.

2. Dissemination:

When bacteria enter the bloodstream, they can encounter fibrin clots, which can act as barriers.

Streptokinase assists in dissolving these clots, allowing the bacteria to disseminate more widely in the host’s bloodstream and evade the host’s immune response.

*Hyaluronidase:

1. Hydrolysis of Hyaluronic Acid:

Hyaluronidase is an enzyme that hydrolyzes hyaluronic acid, a major component of the extracellular matrix that holds cells together in tissues.

By breaking down hyaluronic acid, hyaluronidase promotes tissue permeability and fluid accumulation, allowing bacteria to more easily penetrate tissues.

2. Tissue Invasion:

When bacteria encounter hyaluronic acid-rich tissues, the action of hyaluronidase can create gaps or spaces in the tissue matrix, enabling the bacteria to invade and spread within host tissues. This enhances their ability to cause tissue damage and establish infection.

Question 22

What is evasion?

State the 4 virulence factors that help in evasion by bacteria.

Describe the mechanisms they use for evasion of the bacteria.

Answer 22

Escaping.

Streptolysin O and S
Streptodornase
C5a peptidase
Streptococcal chemokine protease

1. Streptolysin O and Streptolysin S:

Pore Formation and Cell Lysis: Streptolysin O and Streptolysin S are cytolytic toxins that can form pores in host cell membranes.

This lysis of host cells not only disrupts the host’s immune defenses but also releases bacterial pathogens into the surrounding tissue, allowing them to evade the immune system and spread.

2. Streptodornase (DNase):

Destruction of DNA Traps:
When the host’s immune system detects bacterial invaders, it can release DNA-based traps called neutrophil extracellular traps (NETs) to capture and immobilize bacteria.

Streptodornase (DNase) breaks down these NETs, enabling bacteria to escape the traps, evade capture, and continue to infect host tissues.

3. C5a Peptidase:

Inactivation of Complement:

C5a is a potent pro-inflammatory molecule involved in the host’s immune response.

C5a peptidase cleaves and inactivates C5a, which helps the bacteria avoid the host’s complement system. By reducing the activation of complement, the bacteria can evade the immune response, particularly the recruitment of phagocytes.

4. Streptococcal Chemokine Protease:

Cleavage of Chemokines:

Chemokines are signaling molecules that attract immune cells to sites of infection.

Streptococcal chemokine protease can cleave these chemokines, reducing their ability to recruit immune cells to the infection site. This impairment of immune cell recruitment hinders the host’s ability to mount an effective defense against the infection.

Question 23

State 6 examples of exotoxins of bacteria that act as virulence factors and their effect on the host.

Answer 23

Exotoxins:

1. Diphtheria Toxin (produced by Corynebacterium diphtheriae): This toxin inhibits protein synthesis, leading to tissue damage and symptoms of diphtheria.
2. Botulinum Toxin (produced by Clostridium botulinum): Botulinum toxin is the most potent neurotoxin known. It causes muscle paralysis, leading to botulism.
3. Tetanus Toxin (produced by Clostridium tetani): Tetanus toxin blocks the release of inhibitory neurotransmitters, resulting in muscle spasms and rigidity.
4. Cholera Toxin (produced by Vibrio cholerae): Cholera toxin leads to severe diarrhea and dehydration by overstimulating the secretion of fluid in the intestines.
5. Shiga Toxin (produced by Shigella and certain strains of Escherichia coli): Shiga toxin damages blood vessels and can cause bloody diarrhea, kidney damage, and hemolytic uremic syndrome.
6. Pertussis Toxin (produced by Bordetella pertussis): This toxin impairs the immune system and is associated with whooping cough.

Question 24

State an example of bacteria endotoxin that act as virulence factors and their effect on the host.

Answer 24

Endotoxins:

Lipopolysaccharide (LPS):

LPS is a component of the outer membrane of Gram-negative bacteria, and it can trigger a strong immune response, leading to inflammation and septic shock.

Question 25

State how the following toxins produced by the Staphylococcus Auren’s affect the host.

Bacillus cereus enterotoxin
Alpha hemolysin

Answer 25

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.

Question 26

State 5 factors that enable differentiation & identification of microorganisms with lab tests.

Answer 26

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

Question 27

Who devised Gram stain?

What was the reason?

Answer 27

Devised by Danish bacteriologist, Hans Christian Gram, 1882 (published in 1884).

To discriminate between pneumococci & Klebsiella pneumoniae in lung tissue.

Question 28

Describe Gram staining principle & procedure.

Answer 28

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

Question 29

Besides the Gram stain, there are a wide range of other staining methods available, for instance:

Answer 29

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.

Question 30

Mycobacteria have a thick cell wall.

How many % of lipids does it contain?

Why are mycobacterium impermeable to most staining including Gram staining (Nocardia & Rhod coccus)?

Answer 30

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)

Question 31

State the stain used for mycobacteria.

What are its Primary and secondary stains?

Answer 31

So Acid fast stain is used:

Carbolfuchsin (primary stain)
Methylene blue (counter stain)

Question 32

Describe Ziehl-Nelsen staining Principle & procedure. (Acid fast stain)

Answer 32

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.

Question 33

State the Gram-positive tests.

Answer 33

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

Question 34

What is catalase test?

What is the function of catalase enzyme?

Answer 34

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.

Question 35

Describe how the catalase test is done and state its positive results.

What Gram -positive organisms are catalase positive?

Answer 35

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.

Question 36

What is hemolysis?

Describe the three main types of hemolysis observed on blood agar plates.

Answer 36

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.

Question 37

Classify Gram-positive bacteria under alpha, beta and gamma hemolysis.

Answer 37

Alpha () hemolysis
Streptococcus pneumoniae
viridians Streptococcus spp

Beta () hemolysis
Streptococcus pyogenes
S. agalactiae (sometimes-

Gamma () hemolysis
-Enterococcus spp
-Streptococcus Bovis

Question 38

β-Hemolytic Streptococcus spp. are further classified into “Lancefield groups(A-H, K-V)”, based on “C” carbohydrate in the cell wall & its reactions to diff. antisera.

Groups A, B, C, D & G most commonly associated with human infections. Give examples.

Answer 38

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

Question 39

Name a test used in differentiating amongst β-hemolytic Streptococcus spp.

Answer 39

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).

Question 40

Name a test used in differentiation of α-hemolytic Streptococcus pneumoniae from another α-hemolytic Streptococcus spp.

Answer 40

Bile (sodium deoxycholate) solubility test

Question 41

Describe how the bile Solubility test is done and State its positive results.

Answer 41

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.

Question 42

Name an autolytic enzyme, an amidase triggering natural autolysis, in presence of bile salts in S. pneumoniae when using bile solubility test.

Answer 42

S. pneumoniae expresses an autolytic enzyme, an amidase triggering natural autolysis, in presence of bile salts.

Whereas other α- hemolytic Streptococci do not lyse.

Question 43

Describe the principle of lysis of DNA test.

Answer 43

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.

Question 44

Why add HCL in the lysis of DNA
test?

Answer 44

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.

Question 45

What is Clotting of plasma - Tube coagulase test?

What is the function of coagulase enzyme?

Answer 45

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.

Question 46

Describe the procedure for performing a coagulase test.

What is the positive result supposed to be like?

Answer 46

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.

Question 47

Natural/ innate resistance to certain antibiotics as a differential factor.

What is Novobiocin resistance?

Describe the MOA of Novobiocin antibiotic.

Describe what the 2 ranges of the zone of inhibition mean.

Answer 47

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. saprophyticusis 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.

Question 48

Outline ways in which Novobiocin resistance comes about.

Answer 48

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.

Question 49

What is the principle of the Optochin Test?

What is it used for?

Describe the procedure.

How do positive results look like?

Answer 49

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.

Question 50

Susceptible:Zone of inhibition >14 mm
Presumptive positive for S. pneumoniae.
Resistant:No zone of inhibition

What test is this?

Answer 50

Optochin test

Question 51

What is the principle of Bacitracin test?

Answer 51

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.

Question 52

Describe the procedure and positive results for Bacitracin test.

Answer 52

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.

Question 53

Susceptible:Zone of inhibition >10 mm
POS: any zone of inhibition shows bacterium susceptibleNEG:No zone of inhibition shows bacterium is resistant.

What test is this?

Answer 53

Bacitracin test

Question 54

What is Litmus milk test (Gram+ve & Gram–ve bacteria)?

What components are contained in milk?

State the color for litmus paper in alkaline and acidic condition.

Answer 54

“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.

Question 55

Describe what the following results mean in litmus milk test.

1. Change in Color:

-Purple/Pink:
-Light Blue:
-Yellow/Clear:

2. Clot Formation:

Curds or Clots:

3. Gas Production:

. Gas Bubbles:

4. Protein Digestion:

Peptization:

5. Ejection and Sediment Formation:

-Whey Separation and Sediment:

Answer 55

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.

Question 56

What is the largest heterogenous Gram-positive rods/bacilli family called?

Answer 56

Enterobacteriaceae largest heterogenous Gram-positive rods/bacilli family.

At times referred to as “enterics” as they are commensals of human colons.

Question 57

Certain Enterobacteriaceae spp. are common pathogens of human infections i.e.

Answer 57

Salmonella spp.

Shigella spp.

Escherichia coli

Klebsiella spp

Proteus spp.

Yersinia pestis

Question 58

Differentiation Gram -negative by fermentation of carbohydrates or sugars

Carbohydrates as the main energy source of many bacteria.

How are they metabolized?

What are the products?

Name Sugars/ carbohydrates typically used in laboratory media incl. differential media.

Answer 58

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

Question 59

Describe the procedure for Differentiation by fermentation of carbohydrates.

Answer 59

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.

Question 60

Enterobacteriaceae spp. ferment glucose but fermentation of other sugars varies e.g. lactose. Lactose fermentation is used for differentiation of enteric bacteria.

What are lactose fermenting Enterobacteriaceae called?

Outline them.

What are non-lactose fermenting Enterobacteriaceae called?
Outline them.

Answer 60

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

Question 61

Culture of Enterobacteriaceae species

Suspected Enterobacteriaceae spp. are initially grown on culture media: “enriched non-selective” media & “selective+differential” media.

State 2 examples of “Enriched non-selective” media.

Choc. agar is essentially blood agar but comprises _____________, giving it a chocolate-brown color.

Answer 61

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 & Hemophilusspp. are grown on Choc. Agar because they can’t grow in blood agar.

Question 62

Selective & differential media for enteric bacteria

What are Selective & differential media?

What are enteric bacteria?
Give 3 examples of enteric bacteria.

Answer 62

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.

Question 63

Describe how Mackonkey agar is a selective & differential media for enteric bacteria.

Describe how Eosin Methylene blue agar is a selective & differential media for enteric bacteria.

Answer 63

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

Question 64

Biochemical tests for Gram-negative bacteria.

Outline tests used to identify Gram-negative bacteria.

Answer 64

*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

Question 65

What is the oxidase test used to differentiate which groups of microorganisms?

Describe the principle of oxidase test.

What is the positive result for the test?

Answer 65

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 cytochromecwhile reducing O2 to H2O

For the oxidase test, reagent tetra-methyl-p-phenylenediamine dihydrochloride, is an artificial electron donor for cytochromec.

When the reagent is oxidized by cytochromec, it changes from colorless to a dark blue or purple compound.

Question 66

What organisms are negative for the oxidase test?

Give example of positive organisms for oxidase test.

Answer 66

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.

Question 67

What Gram-positive cocci can oxidase test be used to distinguish?

Answer 67

Oxidase test can also be used to distinguish Gram-positive cocci:

-Neisseria gonorrhoeae (oxidase +ve)
-Staphylococcus spp. & Streptococcus spp. (oxidase -ve).

Question 68

IMViC tests is a set of four biochemical tests commonlyused to differentiate Enterobacteriaceae species.

Capital letters ‘IMViC’ refer to the four tests.
State them.

Answer 68

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

Question 69

Describe the principle of Indole test.

Name the media used for the indole test.

Interpret positive and negative results.

Answer 69

Principle: to determine if bacteriumcan break down amino acid ‘tryptophan’ by action of tryptophanase intopyruvic 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

Question 70

Describe the principle of methyl red test.

What are the positive result and negative results?

Answer 70

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.

Question 71

Describe the principle of the VOGES-PROSKAUER-VP.

Interpret the result.

Answer 71

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.

Question 72

What can one achieve by combination of the Voges-Proskauer test with the Methyl Red test?

Answer 72

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.

Question 73

What is the principle of the citrate test?

Interprete the results.

Answer 73

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.

Question 74

SIM agar is semi-solid. It’s typically prepared in tubes referred to as stabs or deeps (not slanted).

State the 3 components found in SIM agar.

It’s used for three tests simultaneously.
*State the 3 tests done using SIM agar.
*What are their positive results.

How are bacteria inoculated into the medium?

Answer 74

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.

Question 75

Describe the principle of the Tripple Sugar Iron (TSI) agar.

Describe the procedure for TSI test.

Interpret the results.

Answer 75

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.

Question 76

Used to differentiate Enterobacteriaceae species, based on:

Carbohydrate fermentation
TSI contains three sugars. State the 3 sugars and their percentages.

Answer 76

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 sulfateorferrous 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

Question 77

If either sucrose or lactose are fermented - sufficient acid production turns pH indicator yellow in both butt& the slant (A/A). If gas is formed, shows as bubbles in butt/ cracking or separation of agar (A/A,G)

Only glucose is fermented - acid production turns butt yellow but insufficient acid will not affect the pH indicator in the slant, thus remains red (gas may be produced) (K/A or K/A,G)

If the bacterium is able to also generate H2S, it reacts with iron to form ferrous sulfide, black precipitate (K/A,H2S)

Minimal to no fermentation of any of the sugars - the slant& butt remain red (K/K)

The following results are for which test?

Answer 77

TSI: Tripple Sugar Iron test

Question 78

Describe the principle of the urease test.

Answer 78

Differentiate Enterobacteriaceae species by determining the ability (or lack of) to hydrolyzeureaproducing ammonia&CO2 , by the activity of urease.

Formation ofammoniaalkalinizesthe medium & changes the pH causing the pH indicator to change color fromlight orangeat todeep pink.

Question 79

Tests used in investigating bacteria for susceptibility to antibiotics.

Describe the principle of the Kirby-Bauer test.

Answer 79

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.

Question 80

Describe the procedure of the Kirby-Bauer test.

Answer 80

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.

Question 81

Interpret the results for the Kirby-Bauer (KB) test.

Answer 81

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.

Question 82

Outline limitations of Kirby-Bauer test.

State 4 factors that can affect the zone of inhibition size.

Answer 82

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

Question 83

Describe the procedure for anti-microbial dilution test as an anti-microbial susceptibility test.

Interpret the results.

Answer 83

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.

Question 84

Antimicrobial dilution tests used to determine:

*Minimal Inhibitory Concentration (MIC)

*Minimal Bactericidal Concentration (MBC)

What is the importance of determining MIC & MBC?

Answer 84

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.

Question 85

The lowest dilution of the antimicrobial that inhibits cloudiness (turbidity) is the _________.

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 _____________.

Answer 85

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).

Question 86

What are alternative tests?
Describe the procedure.
What does E-test stand for?
Why do the alternative test?

Answer 86

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 asEpsilometer 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.

Question 87

Can the MBC be determined from an E-test?

Answer 87

The MBC cannot be determined from the E-test.