L4 CLIN BACTE - Bacterial Identification

Bacterial Identification

↳ Systematic approach to identifying bacterial species.

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↳ Critical for clinical diagnosis and research.

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↳ Combines morphological, biochemical, and molecular methods.

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↳ Essential for proper treatment selection and research applications.

Systematic

research

molecular

treatment

BACTERIAL IDENTIFICATION:

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↳ _______________ approach to identifying bacterial species.

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↳ Critical for clinical diagnosis and _________.

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↳ Combines morphological, biochemical, and _______________ methods.

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↳ Essential for proper ______________ selection and research applications.

Colony Morphology Assessment

↳ First-line identification method.

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↳ Key characteristics observed:

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…..➙ Hemolysis patterns

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…..➙ Pigmentation

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…..➙ Colony size and texture

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…..➙ Adherence to agar

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…..➙ Surface characteristics

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…..➙ Opacity (transparent, translucent, opaque)

First

Hemolysis

texture

Adherence

Surface

Opacity

COLONY MORPHOLOGY ASSESSMENT:

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↳ _______-line identification method.

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↳ Key characteristics observed:

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…..➙ _____________ patterns

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…..➙ Pigmentation

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…..➙ Colony size and _____________

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…..➙ _________________ to agar

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…..➙ ____________ characteristics

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…..➙ _____________ (transparent, translucent, opaque)

MaC Conkey Agar

↳ Differential = Lactose fermenter bacteria

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↳ Selective = Gram (-) bacteria

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…..➙ Escherichia coli

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…..➙ Enterobacter aerogenes

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…..➙ Klebsiella pneumoniae

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…..➙ Proteus vulgaris

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…..➙ Salmonella typhimurium

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…..➙ Staphylococcus aureus

Lactose

-

aerogenes

vulgaris

typhimurium

MAC CONKEY AGAR:

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↳ Differential = ___________ fermenter bacteria

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↳ Selective = Gram (___) bacteria

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…..➙ Escherichia coli

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…..➙ Enterobacter ________________

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…..➙ Klebsiella pneumoniae

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…..➙ Proteus ______________

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…..➙ Salmonella __________________

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…..➙ Staphylococcus aureus

Eosin Methylene Blue (EMB) Agar

↳ Slightly selective for gram-negative bacteria.

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↳ Differential medium, distinguish lactose fermenters.

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↳ Escherichia coli has a distinct green metallic sheen appearance.

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↳ Enterobacter aerogenes is a a lactose fermenter -> acid production -> dark colored colonies.

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↳ Proteus vulgaris, Salmonella typhimurium and Staphylococcus aureus are non-lactose fermenters -> deaminate CHON -> alkaline -> colorless colonies.

negative

Differential

green

dark

alkaline

EOSIN METHYLENE BLUE (EMB) AGAR:

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↳ Slightly selective for gram-______________ bacteria.

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↳ ________________ medium, distinguish lactose fermenters.

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↳ Escherichia coli has a distinct ___________ metallic sheen appearance.

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↳ Enterobacter aerogenes is a a lactose fermenter -> acid production -> _________ colored colonies.

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↳ Proteus vulgaris, Salmonella typhimurium and Staphylococcus aureus are non-lactose fermenters -> deaminate CHON -> ______________ -> colorless colonies.

Hectoen Enteric Agar

↳ A selective and differential agar primarily used to recover Salmonella spp. and Shigella spp.

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↳ Has indicators for lactose fermentation and H2S production.

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↳ Contains various sugars (lactose, sucrose, salicin) which are not utilized by Salmonella and Shigella (only utilized by lactose enterics). Also contains peptone, which the non-lactose fermenters prefer.

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↳ Also has thiosulfate/ferric ammonium citrate which produce a black precipitate in the presence of H2S, distinguishing Salmonella vs Shigella.

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↳ 2 color changes (Lactose/Peptone utilization):

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…..➙ Lactose -> acidify > red/yellow (e.g. E. coli)

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…..➙ Peptone -> alkalinize -> blue agar (Salmonella and Shigella).

…..

↳ H2S production:

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…..➙ (-) H2S -> green colonies -> Shigella

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…..➙ (+) H2S -> black colonies -> Salmonella

selective

fermentation

salicin

peptone

ferric

black

Shigella

HECTOEN ENTERIC AGAR:

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↳ A ____________ and differential agar primarily used to recover Salmonella spp. and Shigella spp.

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↳ Has indicators for lactose _________________ and H2S production.

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↳ Contains various sugars (lactose, sucrose, __________) which are not utilized by Salmonella and Shigella (only utilized by lactose enterics). Also contains _____________, which the non-lactose fermenters prefer.

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↳ Also has thiosulfate/_________ ammonium citrate which produce a ___________ precipitate in the presence of H2S, distinguishing Salmonella vs _____________.

yellow

Peptone

blue

-

Shigella

black

HECTOEN ENTERIC AGAR:

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↳ 2 color changes (Lactose/Peptone utilization):

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…..➙ Lactose -> acidify > red/_____________ (e.g. E. coli)

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…..➙ _____________ -> alkalinize -> _________ agar (Salmonella and Shigella).

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↳ H2S production:

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…..➙ (__) H2S -> green colonies -> ____________

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…..➙ (+) H2S -> _________ colonies -> Salmonella

Gram Positive Bacteria

GRAM STAINING RESPONSE:

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↳ Have a thick peptidoglycan layer in their cell walls, which retains the purple dye of the Gram stain.

thick

purple

GRAM POSITIVE BACTERIA:

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↳ Have a _________ peptidoglycan layer in their cell walls, which retains the ___________ dye of the Gram stain.

Gram Negative Bacteria

GRAM STAINING RESPONSE:

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↳ Have a thinner peptidoglycan layer that loses the purple dye and takes on the counterstain color.

thinner

counterstain

GRAM NEGATIVE BACTERIA:

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↳ Have a ____________ peptidoglycan layer that loses the purple dye and takes on the __________________ color.

Biochemical Tests

↳ Analyzes metabolic activities, enzyme production, and substrate utilization to identify microorganisms.

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↳ These tests differentiate between bacteria based on their unique biochemical properties and are used for various purposes, including identifying pathogenic organisms, distinguishing between species, and monitoring treatment.

metabolic

substrate

biochemical

species

BIOCHEMICAL TESTS:

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↳ Analyzes _______________ activities, enzyme production, and _____________ utilization to identify microorganisms.

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↳ These tests differentiate between bacteria based on their unique __________________ properties and are used for various purposes, including identifying pathogenic organisms, distinguishing between ___________, and monitoring treatment.

Metabolic Activity

HOW BIOCHEMICAL TESTS WORK:

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↳ The tests are based on the metabolic capabilities of microorganisms. Bacteria have inherent properties, such as the ability to produce enzymes or ferment specific substrates, which can be detected through these tests.

capabilities

enzymes

substrates

METABOLIC ACTIVITY:

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↳ The tests are based on the metabolic ________________ of microorganisms. Bacteria have inherent properties, such as the ability to produce _____________ or ferment specific _____________, which can be detected through these tests.

Enzyme Presence

HOW BIOCHEMICAL TESTS WORK:

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↳ Tests identify the presence or absence of specific enzymes, such as catalase, which decomposes hydrogen peroxide.

absence

peroxide

ENZYME PRESENCE:

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↳ Tests identify the presence or ______________ of specific enzymes, such as catalase, which decomposes hydrogen ______________.

Substrate Utilization

HOW BIOCHEMICAL TESTS WORK:

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↳ The ability of bacteria to utilize different carbohydrates and other compounds as energy sources is also tested.

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↳ Different bacteria will produce different byproducts when they ferment or metabolize these substrates.

carbohydrates

energy

byproducts

substrates

SUBSTRATE UTILIZATION:

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↳ The ability of bacteria to utilize different ____________________ and other compounds as ___________ sources is also tested.

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↳ Different bacteria will produce different _______________ when they ferment or metabolize these _____________.

Catalase Test

Coagulase Test

Oxidase Test

Triple Sugar Iron (TSI) Agar Test

IMViC Test

Indole Test

Methyl Red Test

Voges-Proskauer

Citrate Test

Lysine Decarboxylase Test

Urease Test

Lactose Fermentation Test

Motility Test

14 Types of Biochemical Tests

Catalase Test

TYPE OF BIOCHEMICAL TESTS:

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↳ Purpose = differentiates ***-positive from ***-negative species.

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↳ Mechanism = detects H202 breakdown to water and oxygen.

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↳ Key reactions:

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…..➙ Positive = Copious bubble formation.

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…..➙ Negative: No/few bubbles.

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↳ Common applications:

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…..➙ Distinguishing Staphylococci (+) from Streptococci (-).

negative

H202

oxygen

Copious

Streptococci

CATALASE TEST:

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↳ Purpose = differentiates catalase-positive from catalase-_____________ species.

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↳ Mechanism = detects _________ breakdown to water and __________.

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↳ Key reactions:

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…..➙ Positive = ____________ bubble formation.

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…..➙ Negative: No/few bubbles.

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↳ Common applications:

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…..➙ Distinguishing Staphylococci (+) from ___________________ (-).

Coagulase Test

TYPE OF BIOCHEMICAL TESTS:

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↳ *** enzyme = produced by certain bacteria, especially S. aureus.

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↳ Mechanism = *** reacts with a plasma protein called prothrombin, converts fibrinogen into fibrin.

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↳ Result = Fibrin forms a visible clot in the plasma, mimicking blood coagulation.

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↳ Methods:

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…..➙ Slide test = clumping factor and is detected rapidly.

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…..➙ Tube test = clot formation.

S. aureus

prothrombin

fibrin

clot

coagulation

Slide

clumping

Tube

clot

COAGULASE TEST:

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↳ Coagulase enzyme = produced by certain bacteria, especially ____________.

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↳ Mechanism = coagulase reacts with a plasma protein called ___________________, converts fibrinogen into _________.

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↳ Result = Fibrin forms a visible ______ in the plasma, mimicking blood ___________________.

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↳ Methods:

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…..➙ _________ test = ______________ factor and is detected rapidly.

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…..➙ ________ test = _______ formation.

Oxidase Test

TYPE OF BIOCHEMICAL TESTS:

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↳ Purpose = identifies cytochrome *** activity.

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↳ Method = Kovac's procedure

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↳ Results interpretation:

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…..➙ Positive = dark purple color within 10 seconds.

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…..➙ Negative = no color change.

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↳ Key example:

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…..➙ Pseudomonas aeruginosa (+)

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…..➙ Escherichia coli (-)

cytochrome

Kovac

purple

10

aeruginosa

Escherichia

OXIDASE TEST:

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↳ Purpose = identifies _________________ oxidase activity.

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↳ Method = _________'s procedure

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↳ Results interpretation:

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…..➙ Positive = dark ___________ color within ___ seconds.

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…..➙ Negative = no color change.

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↳ Key example:

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…..➙ Pseudomonas _________________ (+)

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…..➙ _________________ coli (-)

Triple Sugar Iron (TSI) Agar Test

TYPE OF BIOCHEMICAL TESTS:

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↳ Used to identify and distinguish Enterobacteriaceae by their ability to catabolize lactose, sucrose, or glucose and to liberate sulfides.

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↳ Contains 1% lactose and sucrose, 0.1% glucose, ferrous ammonium sulfate, sodium thiosulfate and phenol red.

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↳ Phenol red:

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…..➙ pH indicator

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…..➙ Detects acid production from carbohydrate fermentation.

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…..➙ Fermentation -> acid formation -> turns yellow.

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…..➙ K = alkaline; a = acid.

bacteriaceae

sucrose

sulfides

0.1

ferrous

thiosulfate

pH

carbohydrate

yellow

alkaline

TRIPLE SUGAR IRON (TSI) AGAR TEST:

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↳ Used to identify and distinguish Entero_______________ by their ability to catabolize lactose, ____________, or glucose and to liberate ___________.

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↳ Contains 1% lactose and sucrose, ____% glucose, ___________ ammonium sulfate, sodium _______________ and phenol red.

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↳ Phenol red:

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…..➙ ____ indicator

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…..➙ Detects acid production from ____________________ fermentation.

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…..➙ Fermentation -> acid formation -> turns ___________.

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…..➙ K = _____________; a = acid.

K/A

UNDER TRIPLE SUGAR IRON (TSI) AGAR TEST:

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↳ Red slant / Yellow butt

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…..➙ Interpretation = glucose fermentation.

Red

glucose

K/A:

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↳ _______ slant / Yellow butt

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…..➙ Interpretation = ___________ fermentation.

A/A

UNDER TRIPLE SUGAR IRON (TSI) AGAR TEST:

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↳ Yellow slant / Yellow butt

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…..➙ Interpretation = glucose and lactose/sucrose fermentation.

butt

lactose

A/A:

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↳ Yellow slant / Yellow _______

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…..➙ Interpretation = glucose and ____________/sucrose fermentation.

(+) H2S

UNDER TRIPLE SUGAR IRON (TSI) AGAR TEST:

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↳ Formation = blackening of agar.

blackening

(+) H2S:

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↳ Formation = _______________ of agar.

(+) Gas

UNDER TRIPLE SUGAR IRON (TSI) AGAR TEST:

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↳ Formation = splitting of agar.

splitting

(+) GAS:

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↳ Formation = _____________ of agar.

IMViC Test

TYPE OF BIOCHEMICAL TESTS:

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↳ Set of four biochemical tests:

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…..➙ Indole

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…..➙ Methyl Red

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…..➙ Voges-Proskauer

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…..➙ Citrate

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↳ Used to differentiate members of the Enterobacteriaceae family, especially Escherichia coli and Enterobacter/Klebsiella species.

Methyl

Proskauer

family

Klebsiella

IMViC TESTS:

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↳ Set of four biochemical tests:

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…..➙ Indole

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…..➙ ___________ Red

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…..➙ Voges-_____________

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…..➙ Citrate

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↳ Used to differentiate members of the Enterobacteriaceae __________, especially Escherichia coli and Enterobacter/_____________ species.

Indole Test

TYPE OF BIOCHEMICAL TESTS:

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↳ Detects the enzyme tryptophanase, which breaks down tryptophan into ***.

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↳ Positive result = red ring after adding Kovac's reagent.

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…..➙ Example: E. coli and P. vulgaris is indole-positive.

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↳ Negative result = yellow ring.

tryptophanase

red

reagent

vulgaris

INDOLE TEST:

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↳ Detects the enzyme _____________________, which breaks down tryptophan into indole.

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↳ Positive result = _____ ring after adding Kovac's ________________.

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…..➙ Example: E. coli and P. ___________ is indole-positive.

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↳ Negative result = yellow ring.

Methyl Red Test

TYPE OF BIOCHEMICAL TESTS:

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↳ Detects mixed acid fermentation of glucose.

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↳ After glucose is fermented to pyruvate, an organism takes two pathways:

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…..➙ Mixed-acid

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…..➙ Stable acids

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…..➙ pH below 4.4

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↳ Positive result = red color after adding methyl red indicator.

glucose

pyruvate

Stable

4.4

methyl

METHYL RED TEST:

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↳ Detects mixed acid fermentation of ___________.

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↳ After glucose is fermented to ______________, an organism takes two pathways:

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…..➙ Mixed-acid

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…..➙ ___________ acids

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…..➙ pH below _____

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↳ Positive result = red color after adding __________ red indicator.

Voges-Proskauer

TYPE OF BIOCHEMICAL TESTS:

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↳ Detects mixed acid fermentation of glucose.

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↳ After glucose is fermented to pyruvate, an organism takes two pathways:

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…..➙ 2 Butanediol = neutral acetoin.

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…..➙ 2,3-butanediol = spares the pH.

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↳ Positive = pink or red color at the surface of the medium (acetoin present) Alpha-Naphthol.

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↳ Negative = yellow or copper color at the surface of the medium (acetoin absent).

mixed

pyruvate

acetoin

2,3

pH

red

surface

Naphthol

copper

VOGES-PROSKAUER:

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↳ Detects __________ acid fermentation of glucose.

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↳ After glucose is fermented to ______________, an organism takes two pathways:

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…..➙ 2 Butanediol = neutral _______.

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…..➙ ____-butanediol = spares the ____.

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↳ Positive = pink or ______ color at the ___________ of the medium (acetoin present) Alpha-_______________.

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↳ Negative = yellow or ___________ color at the surface of the medium (acetoin absent).

Citrate Test

TYPE OF BIOCHEMICAL TESTS:

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↳ Determine the ability of the organism to utilize sodium citrate as its only carbon source & inorganic ammonium as its nitrogen source.

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↳ Bacteria turn the Bromothymol blue indicator from green to blue.

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↳ Positive = blue.

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…..➙ Memory aid = Citrate -> Cyan = blue.

citrate

ammonium

Bromothymol

green

blue

CITRATE TEST:

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↳ Determine the ability of the organism to utilize sodium __________ as its only carbon source & inorganic ______________ as its nitrogen source.

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↳ Bacteria turn the ____________________ blue indicator from __________ to blue.

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↳ Positive = blue.

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…..➙ Memory aid = Citrate -> Cyan = _______.

Lysine Decarboxylase Test

TYPE OF BIOCHEMICAL TESTS:

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↳ Detects whether bacteria can produce the enzyme lysine.

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↳ Lysine breaks down into cadaverine and carbon dioxide.

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↳ Positive result = turns the medium purple (alkaline).

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↳ Negative result = remains yellow (acidic).

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↳ Useful for differentiating Salmonella (positive) from Shigella (negative).

lysine

cadaverine

purple

alkaline

Salmonella

LYSINE DECARBOXYLASE TEST:

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↳ Detects whether bacteria can produce the enzyme _________.

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↳ Lysine breaks down into _________________ and carbon dioxide.

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↳ Positive result = turns the medium ________ (____________).

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↳ Negative result = remains yellow (acidic).

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↳ Useful for differentiating ______________ (positive) from Shigella (negative).

Urease Test

TYPE OF BIOCHEMICAL TESTS:

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↳ Used to determine whether bacteria produce the enzyme ***.

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↳ Hydrolyzes urea into ammonia (NH3) and carbon dioxide (CO2).

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↳ The pH indicator phenol red changes color:

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…..➙ Yellow/orange = acidic (negative).

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…..➙ Pink/magenta = alkaline (positive).

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↳ Useful for identifying Proteus species, which are rapid *** producers.

urease

ammonia

dioxide

phenol

acidic

magenta

Proteus

UREASE TEST:

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↳ Used to determine whether bacteria produce the enzyme _________.

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↳ Hydrolyzes urea into _____________ (NH3) and carbon __________ (CO2).

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↳ The pH indicator ___________ red changes color:

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…..➙ Yellow/orange = __________ (negative).

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…..➙ Pink/____________ = alkaline (positive).

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↳ Useful for identifying ___________ species, which are rapid urease producers.

Lactose Fermentation Test

TYPE OF BIOCHEMICAL TESTS:

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↳ Used to determine whether bacteria can ferment *** to produce acid (and sometimes gas).

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↳ Principle:

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…..➙ Bacteria ferment *** -> produce acid (lowers pH) + gas.

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…..➙ Acid production changes the color of the pH indicator (phenol red or bromothymol blue).

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↳ Gas production is detected by a Durham tube (small inverted tube inside the broth).

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…..➙ Positive = yellow broth (acid) + gas bubble in Durham tube.

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…..➙ Negative = red/orange broth (no acid) and no gas.

acid

lowers

bromothymol

Durham

yellow

bubble

orange

LACTOSE FERMENTATION TEST:

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↳ Used to determine whether bacteria can ferment lactose to produce _____ (and sometimes gas).

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↳ Principle:

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…..➙ Bacteria ferment lactose -> produce acid (___________ pH) + gas.

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…..➙ Acid production changes the color of the pH indicator (phenol red or _________________ blue).

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↳ Gas production is detected by a ____________ tube (small inverted tube inside the broth).

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…..➙ Positive = ___________ broth (acid) + gas __________ in Durham tube.

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…..➙ Negative = red/__________ broth (no acid) and no gas.

Motility Test

TYPE OF BIOCHEMICAL TESTS:

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↳ Used to determine whether bacteria are capable of motility, usually through flagella.

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↳ It helps distinguish motile organisms (like E. coli) from non-motile ones (like Klebsiella pneumoniae).

flagella

Klebsiella

MOTILITY TEST:

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↳ Used to determine whether bacteria are capable of motility, usually through _____________.

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↳ It helps distinguish motile organisms (like E. coli) from non-motile ones (like _____________ pneumoniae).

DNA-DNA Hybridization

16S rRNA Gene Sequencing

Whole Genome Sequencing

3 Genotypic Methods

DNA-DNA Hybridization

GENOTYPIC METHODS:

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↳ Measures genomic similarity by assessing the extent of strand re-annealing between DNA of different organisms, aiding species delineation in microbial taxonomy.

similarity

annealing

delineation

DNA-DNA HYBRIDIZATION:

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↳ Measures genomic ____________ by assessing the extent of strand re-______________ between DNA of different organisms, aiding species _________________ in microbial taxonomy.

16S rRNA Gene Sequencing

GENOTYPIC METHODS:

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↳ Identifies and classifies microbes by analyzing conserved genetic regions, enabling precise phylogenetic placement and differentiation at the species level.

regions

phylogenetic

species

16S RRNA GENE SEQUENCING:

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↳ Identifies and classifies microbes by analyzing conserved genetic __________, enabling precise _________________ placement and differentiation at the __________ level.

Whole Genome Sequencing

GENOTYPIC METHODS:

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↳ Analyzes the entire genetic material of microbes, providing high-resolution data for precise identification, evolutionary relationships, and functional insights, revolutionizing microbial taxonomy and phylogenetic studies.

microbes

resolution

insights

studies

WHOLE GENOME SEQUENCING:

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↳ Analyzes the entire genetic material of ____________, providing high-_______________ data for precise identification, evolutionary relationships, and functional ____________, revolutionizing microbial taxonomy and phylogenetic ___________.