Bacteriology 2: Methods of Laboratory Diagnosis of Bacterial Diseases

Conventional bacterial diagnosis

process of isolating, identifying, and characterizing bacteria using culture, microscopy, biochemical testing, and antibiotic susceptibility testing

Five major steps of conventional bacterial diagnosis:

1.) Identify suspected pathogens

2.) Collect and submit specimens

3.) Culture and isolate bacteria

4.) Identify bacterial species

5.) Perform antibiotic susceptibility testing

Six factors that help veterinarians identify suspected bacterial pathogens

1.) Animal species

2.) Affected organ system

3.) Clinical history and signs

4.) Epidemiology

5.) Gross pathology

6.) Knowledge of veterinary bacteriology

What types of specimens can be submitted for bacterial diagnosis?

Blood, feces, and tissue biopsies

bacterial culture

process of growing bacteria under controlled laboratory conditions to isolate and identify them

Two major requirements for bacterial culture:

1.) Oxygen requirements

2.) Nutritional requirements

Three classifications of bacteria based on oxygen requirements:

1.) Obligate aerobes

2.) Facultative anaerobes

3.) Obligate anaerobes

Obligate aerobes

Bacteria that require oxygen for growth

Facultative anaerobes

Bacteria that can grow with or without oxygen

Obligate anaerobes

Bacteria that cannot tolerate oxygen and may be killed by exposure to it

Why must obligate anaerobes be protected from oxygen during specimen collection and transport?

Exposure to oxygen can kill these bacteria, preventing successful culture

What is an anaerobic jar?

A laboratory container that creates an oxygen-free environment for culturing anaerobic bacteria

Two major components used to remove oxygen from an anaerobic jar:

1.) Hydrogen gas generator

2.) Palladium or platinum catalyst

How does an anaerobic jar create an oxygen-free environment?

Hydrogen reacts with oxygen on the catalyst surface, consuming oxygen and creating anaerobic conditions

What is culture media?

Nutrient-containing material used to support bacterial growth in the laboratory

Two major types of bacterial culture media:

1.) Nonselective media

2.) Selective media

Nonselective media

Culture media that support the growth of many different bacterial species

Selective media

Culture media that contain substances that inhibit certain bacteria while allowing others to grow

What is an example of nonselective culture media?

Blood agar

Why is blood agar useful when the suspected bacterial pathogen is unknown?

It supports the growth of many bacterial species and allows evaluation of colony characteristics and hemolysis

Four colony characteristics evaluated on blood agar:

1.) Diameter

2.) Elevation

3.) Colony margin

4.) Hemolysis

Two major types of hemolysis evaluated on blood agar:

1.) Alpha hemolysis

2.) Beta hemolysis

Alpha hemolysis

Partial hemolysis of red blood cells

Beta hemolysis

Complete hemolysis of red blood cells

Two selective agar media used for bacterial identification:

1.) MacConkey agar

2.) Mannitol salt agar

MacConkey agar

Selective and differential culture medium used to isolate enteric bacteria and distinguish lactose fermenters

What selective ingredient is found in MacConkey agar?

Bile salts

Why are bile salts used in MacConkey agar?

They inhibit many nonenteric bacteria while allowing bile-tolerant enteric bacteria to grow

Why can enteric bacteria grow on MacConkey agar?

They normally inhabit the intestinal tract and are resistant to bile salts

What differential ingredient is found in MacConkey agar?

Lactose, along with the pH indicator neutral red

What happens when bacteria ferment lactose on MacConkey agar?

Acid is produced, causing colonies to appear red or pink

Mannitol salt agar

Selective and differential culture medium used to isolate staphylococci and distinguish mannitol fermenters

What selective ingredient is found in mannitol salt agar?

7.5% NaCl

Why is a high salt concentration used in mannitol salt agar?

It inhibits many bacteria while allowing salt-tolerant staphylococci to grow

What differential ingredient is found in mannitol salt agar?

Mannitol, along with the pH indicator phenol red

What happens when bacteria ferment mannitol on mannitol salt agar?

Acid is produced, changing the indicator to yellow

Recall the three culture media, their selective components, and their differential properties

1.) Blood agar → Nonselective, differentiates hemolytic reactions

2.) MacConkey agar → Bile salts, differentiates lactose fermentation

3.) Mannitol salt agar → 7.5% NaCl, differentiates mannitol fermentation

What is colony morphology?

The observable physical characteristics of bacterial colonies growing on culture media

What colony appearance is characteristic of Proteus?

Swarming growth that produces a spreading, curled appearance

Why does Proteus produce a spreading colony appearance?

It exhibits swarming motility

What colony appearance may be characteristic of Bacillus and Clostridium?

Filamentous margins resembling kinky hair or a Medusa head

What colony appearance is characteristic of Staphylococcus?

Smooth, shiny colonies

What may a visible zone of hemolysis surrounding a bacterial colony indicate?

The bacterium produces substances capable of lysing red blood cells

Why is colony morphology alone often insufficient for bacterial identification?

Different bacteria may have similar colony appearances and require additional testing

What is bacterial morphology?

The physical characteristics of bacteria, including their shape, size, and arrangement

Three major bacterial cell shapes:

1.) Cocci

2.) Bacilli

3.) Spiral

Three common bacterial cell arrangements:

1.) Chains

2.) Clusters

3.) Pairs

What is Gram staining?

A differential staining technique used to classify bacteria as Gram-positive or Gram-negative

Two classifications of bacteria based on Gram staining:

1.) Gram-positive

2.) Gram-negative

Why is Gram staining useful in bacterial diagnosis?

It helps narrow the possible bacterial species and guides additional diagnostic testing

What is acid-fast staining?

A staining method used to identify bacteria with distinctive acid-fast cell wall properties

What bacterial genus is particularly associated with acid-fast staining?

Mycobacterium

What is bacterial motility?

The ability of bacteria to move actively

Two simple methods for testing bacterial motility:

1.) Hanging-drop method

2.) Soft agar motility test

Hanging-drop method

microscopy technique used to directly observe bacterial movement in a suspended drop of fluid

How is the hanging-drop method performed?

A drop of bacterial suspension is placed on a coverslip, inverted over a depression slide, and examined microscopically

Why is the hanging-drop method useful for determining bacterial motility?

It allows observation of bacterial movement while minimizing interference from air currents

Soft agar motility test

test that determines bacterial motility based on the pattern of growth in semisolid agar

How is the soft agar motility test performed?

Bacteria are introduced into semisolid agar by stab inoculation and observed for growth patterns

What growth pattern indicates nonmotile bacteria in soft agar?

What growth pattern indicates motile bacteria in soft agar?

Growth restricted to the stab line

Diffuse growth spreading away from the stab line

What additional information can the soft agar motility test provide?

Information about bacterial oxygen requirements based on the location of growth

Recall the two motility tests and how motility is identified in each

1.) Hanging-drop method → Direct observation of bacterial movement

2.) Soft agar motility test → Diffuse growth away from the stab line

What are biochemical tests?

Diagnostic tests that identify bacteria based on their metabolic activities or enzyme production

Why are biochemical tests useful in bacterial diagnosis?

They distinguish bacteria that have similar morphology and staining properties

Four major biochemical tests used to distinguish similar bacteria:

1.) Lactose utilization test

2.) Catalase test

3.) Oxidase test

4.) Coagulase test

What is the lactose utilization test?

A biochemical test that determines whether bacteria can use lactose as a carbon and energy source

What type of bacteria are commonly differentiated using lactose utilization?

Gram-negative, facultatively anaerobic enteric rods

Which two bacteria can be differentiated using lactose utilization?

E. coli and Salmonella

Two enzymes involved in bacterial lactose utilization:

1.) Beta-galactoside permease

2.) Beta-galactosidase

What is the function of beta-galactoside permease?

Facilitates lactose transport into the bacterial cell

What is the function of beta-galactosidase?

Breaks down lactose into simpler sugars

Is E. coli lactose-positive or lactose-negative?

Is Salmonella lactose-positive or lactose-negative?

Lactose-positive

Lactose-negative

What reaction occurs when E. coli ferments lactose in MacConkey agar?

Acid production causes red or pink colonies

Why can the lactose utilization test distinguish E. coli from Salmonella?

E. coli can ferment lactose, whereas Salmonella generally cannot

What is the catalase test?

A biochemical test that determines whether bacteria produce the enzyme catalase

Which two bacterial genera are commonly differentiated using the catalase test?

Staphylococcus and Streptococcus

What type of bacteria are Staphylococcus and Streptococcus?

Gram-positive, facultatively anaerobic cocci

What is catalase?

An enzyme that breaks down hydrogen peroxide into water and oxygen

What is the chemical reaction catalyzed by catalase?

2H₂O₂ → 2H₂O + O₂

How is the catalase test performed?

Hydrogen peroxide is applied to a bacterial colony and observed for bubble formation

Why do bubbles form during a positive catalase test? Oxygen gas is released as hydrogen peroxide is broken down

Oxygen gas is released as hydrogen peroxide is broken down

Is Staphylococcus catalase-positive or catalase-negative?

Is Streptococcus catalase-positive or catalase-negative?

Catalase-positive

Catalase-negative

What is observed during a positive catalase test?

What is observed during a negative catalase test?

Gas bubbles form

No gas bubbles form

Why is the catalase test useful for differentiating Staphylococcus and Streptococcus?

Both are Gram-positive cocci, but Staphylococcus produces catalase while Streptococcus generally does not

What is the oxidase test?

A biochemical test that detects bacterial cytochrome oxidase activity in the electron transport chain

What type of bacteria are commonly differentiated using the oxidase test?

Gram-negative bacteria

Which two bacterial families are commonly differentiated using the oxidase test?

Pasteurellaceae and Enterobacteriaceae

What is the role of oxidase in bacterial metabolism?

It participates in the terminal step of the electron transport chain, transferring electrons to oxygen

What reagent is used in the oxidase test?

Tetramethyl-p-phenylenediamine

What is the role of tetramethyl-p-phenylenediamine in the oxidase test?

It acts as an electron donor that changes color when oxidized

What color change indicates a positive oxidase test?

Colorless to blue

What is observed during a negative oxidase test?

No blue color develops

Is Pasteurellaceae oxidase-positive or oxidase-negative?

Is Enterobacteriaceae oxidase-positive or oxidase-negative?

Oxidase-positive

Oxidase-negative

Where are Pasteurellaceae commonly found?

Where are Enterobacteriaceae commonly found?

Oral cavity and respiratory tract

Intestinal tract

Why is the oxidase test useful for differentiating Pasteurellaceae and Enterobacteriaceae?

Both contain Gram-negative, facultatively anaerobic bacteria, but Pasteurellaceae are generally oxidase-positive while Enterobacteriaceae are oxidase-negative

Coagulase test

biochemical test that determines whether bacteria produce the enzyme coagulase

What bacteria are commonly evaluated using the coagulase test?

Staphylococci

Coagulase

enzyme that promotes the conversion of fibrinogen into fibrin, resulting in clot formation

What reaction occurs during a positive coagulase test?

Fibrinogen → Fibrin clot

How is the coagulase test performed?

A bacterial sample is mixed with plasma and observed for coagulation