Foundations - Microbiology

Human microbiota

the microorganisms that naturally inhabit the human body.

Streptococcus species

What are common microbiota found in the oral cavity?

Staphylococcus epidermis and aureus

What are common microbiota found on the skin?

Bacteroides species, Escherichia coli

What are common microbiota found in the large intestine?

Gram-positive cocci

What is the staining and shape of streptococcus and staphylococcus species?

Gram-negative rods

What is the staining and shape of most bacterial species inhabiting the large intestine?

Infectious agents

cause infectious disease, mostly microorganisms, e.g. bacteria, viruses, fungi.

Exudate, pus

What samples are taken for wound infections?

Sputum, blood sample

What samples are taken for pneumonia, i.e. lung infection?

Oropharyngeal swab, bilateral deep nasal swab

What samples are taken for COVID-19?

Mid stream urine sample

What samples are taken for urinary tract infections?

Faecal samples

What samples are taken for gastroenteritis, i.e. stomach infection?

Blood

What samples are taken for bacteraemia, i.e. presence of bacteria in blood stream?

Cerebrospinal fluid (CSF)

What samples are taken for meningitis?

Empirical therapy

the immediate initiation of treatment based on clinical suspicion before definitive lab results identify the specific pathogen.

Before treatment, appropriate specimens, no contamination, proper storage/transport

What are the 4 main principles of specimen collection?

Sputum

a mixture of saliva and mucus, also known as phlegm, that is coughed up from the lower respiratory tract—the lungs and bronchi.

Gram staining

technique used to classify bacteria based on the structure of their cell walls.

Crystal violet

product first applied in gram staining, staining all bacteria purple.

Iodine

product added to form a complex with crystal violet to prevent easy removal of dye during washing.

Decolourisation

washing of bacteria with alcohol or acetone, removing loosely bound stain on gram-negative bacteria.

Thick peptidoglycan layer

Describe the cell wall of gram-positive bacteria which allows them to retain the purple stain during decolourisation.

Thin peptidoglycan layer, outer membrane

Describe the cell wall of gram-negative bacteria which means the purple stain is not retained during decolourisation.

Safranin

product used to counterstain gram-negative bacteria with a pink/red colour.

Catalase test

detects the enzyme which breaks down hydrogen peroxide (H₂O₂) into water and oxygen, observed as bubbles.

Staphylococcus, Streptococcus spp

What are 2 common species which the catalase test differentiates, listed as catalase +, catalase-

Coagulase test

detects the enzyme which causes fibrin clot formation, as bacteria cause clotting of plasma or visible clumping if positive.

Staphylococcus aureus

What staphylococci is positive for the coagulase test?

Oxidase test

detects enzymes involved in oxidative phosphorylation.

Indole test

detects bacteria that produce tryptophanase (breaks down tryptophan to indole).

Carbohydrate fermentation

tests if bacteria can ferment specific sugars (glucose, lactose, etc.) producing acid and/or gas.

Urease test

detects urease production, which hydrolyses urea to ammonia, raising pH.

Motility test

determines if bacteria are motile by tracking movement through semi-solid agar.

Aerobes

organisms that will only grow in the presence of oxygen.

Facultative anaerobes

organisms that will grow in the presence or absence of oxygen.

Obligate anaerobes

organisms that will grow only in the absence of oxygen.

Alpha haemolysis

partial haemolysis by bacteria produces hydrogen peroxide which oxidises haemoglobin to methemoglobin, causing a green haze around colony, e.g. viridans streptococci, Streptococcus pneumoniae.

Beta haemolysis

complete lysis of red blood cells by haemolysins forming a clear zone around the colonies, e.g. Streptococcus pyogenes using streptolysins.

Gamma haemolysis

no haemolysis.

Streptococcus pneumoniae

What is an example of a bacteria that undergoes alpha haemolysis?

Streptococcus pyogenes

What is an example of a bacteria that undergoes beta haemolysis?

Basal media

simple growth media made from meat or yeast infusions, e.g. Nutrient Agar.

Enriched media

selective media have added extra nutrients usually of a complex nature, i.e.

Selective agar

growth medium that contain specific agents that inhibit the growth of some microbes while allowing other to grow.

Differential agar

allows different organisms to be distinguished based on biochemical/metabolic properties. A visible change results e.g. MacConkey agar.

Nutrient Agar (NA)

general-purpose **basal** medium containing peptones, yeast extract, and sodium chloride that supports the growth of many non-fastidious microorganisms.

Blood Agar (SBA or HBA)

a **differential** medium **enriched** with red blood cells (sheep or horse) to support growth of fastidious pathogenic bacteria. Detects haemolysis patterns.

Chocolate Blood Agar (CBA)

an **enriched** blood agar where red blood cells are heated and lysed, releasing growth factors (X and V factors) required by fastidious organisms, e.g. Neisseria gonorrhoeae, Haemophilus influenzae.

Gonococcal Medium

enriched medium like chocolate agar but contains antibiotics allowing selective growth of Neisseria species, particularly Neisseria gonorrhoeae.

Neisseria gonorrhoeae

What is the main species gonococcal medium is selective for?

MacConkey Agar (MAC)

contains bile salts that inhibit many Gram-positive and non-enteric bacteria, making it selective for Gram-negative enteric bacteria. Contains lactose and neutral red indicator to differentiate lactose fermenters.

Pink/red, creamy

What colour are lactose and non-lactose fermenters grown in MacConkey Agar.

Sabouraud Agar (SAB)

selective medium containing high glucose and low pH, which inhibits bacterial growth and allows isolation of fungi and yeasts.

Mannitol Salt Agar (MSA)

contains 7.5% sodium chloride, selecting for salt-tolerant Staphylococcus sp. Contains mannitol and phenol red indicator to differentiate mannitol fermenters.

Yellow, pink/red

What colour are mannitol and non-mannitol fermenters frown in Mannitol Salt Agar?

Staphylococcus aureus

What is a common bacterium that ferments mannitol?

Escherichia coli

What is a common bacterium that ferments lactose?

MALDI-TOF

method for identifying a pathogen based on protein profile of pathogen analysed by spectrometer.

Sequencing genome

method for identifying a pathogen that will provide correct identification, but timely and expensive.

Direct microscopy, bacterial cultivation, molecular methods

What are the 3 main approaches to a new specimen in a diagnostic laboratory?

Direct microscopy

approach to specimen identification, examination of stained and non-stained samples. Provides rapid initial observation to help guide early decisions.

Bacterial cultivation

approach to specimen identification, must be isolated from microbiota and cultured to undergo various methods of identification.

Molecular methods

approach to specimen identification where PCR and serology may be used for direct detection of pathogens

Polymerase chain reaction

rapidly amplifying a DNA fragment that is specific for a pathogen.

Denaturation

(≈95 °C) the double-stranded DNA separates into two single strands.

Annealing

(≈65 °C) short DNA primers bind to complementary sequences on DNA.

Extension

(≈72 °C) DNA polymerase (e.g. Taq polymerase) extends the primers, synthesising new DNA strands.

Gel electrophoresis

technique used after traditional PCR to compare the profile of the target bacterial gene to know bacterial genes.

Quantitative real-time PCR (qPCR)

amplifies specific nucleic acid target and measures the amount of amplified product in real time using fluorescence, negating gel electrophoresis.

Antibiotic sensitivity test

measurement of the susceptibility of bacteria to antibiotics.

Serology

checks for antibodies in the blood, often used for diagnosis of viral infections, can be used for bacteria that can’t be cultured.

IgM antibodies

presence of what kind of antibodies indicate active acute infection, helping distinguish from antibodies present due to past infections?

Enzyme-Linked Immunosorbent Assay (ELISA)

use of specific antibody binding and a colour-producing enzyme reaction to detect presence and quantify antigens or antibodies.

Respiratory chain

pumps H⁺ out of the bacteria cell membrane via proteins.

Proton motive force

electrochemical gradient creates where H⁺ is in a higher concentration outside of the cell.

Cell wall

layer of peptidoglycan surrounding the cell providing shape and rigidity.

Lysis

accumulation of solutes generate 2 atm of pressure, which can easily rupture semi-fluid plasma membrane, so the addition of the cell wall protects from what?

Characteristic shapes

proteins direct synthesis of peptidoglycan layer, determining what?

Peptidoglycan

chains of sugars and short peptides which are crosslinked together to form a lattice-like structure, providing strength.

G, M

in peptidoglycan, the polysaccharide backbone is created from which 2 alternating sugars.

Glycosidic bond

What joins G and M sugars in peptidoglycan?

M

Which sugar do short peptide chains link to in peptidoglycan?

Capsules

thick, tightly packed, polysaccharide or protein layers that cover the cell.

Desiccation, immune detection

What are the 2 main things capsules protect bacteria from?

Adhesion

what is a property that capsules aid in, given their overall negative charge?

Fimbriae

hair-like appendages mainly for adhesion, shorter and thinner than pili.

Pili

hollow fibres with many functions → surface attachment, conjugation, twitching motility.

Flagella

long helical filaments that propel bacteria through aqueous environments.

Surface attachment, conjugation, twitching motility

What are the 3 main roles of pili?

Conjugation

transfer of DNA between bacterial cells.

Twitching motility

crawling motion mediated by the extension, anchoring, and rapid retraction of pili.

Basal body, hook, long filament

What are the 3 components of flagella?

Gram positive

bacteria with a thick peptidoglycan layer anchored to cytoplasmic membrane.

Interbridge

short peptide chain that crosslinking glycan strands providing additional flexibility and added structural rigidity to the peptidoglycan layer for gram positive bacteria.

Teichnoic acids

cell wall polymers composed of glycerol phosphate or ribitol phosphate that bind the layers of peptidoglycan by crosslinking to M sugars.

Lipoteichnoic acids

teichnoic acids with lipid tails that anchor the peptidoglycan layer in the cytoplasmic membrane below.

Gram negative

bacteria with a thin peptidoglycan layer surrounded by a second outer membrane.

Braun lipoproteins

proteins that anchor the outer membrane to the peptidoglycan layer.

Periplasm (peripalsmic space)

space between cytoplasmic membrane and outer membrane.

Outer membrane, Braun lipoproteins, periplasm, lipopolysaccharides

What are the 4 key structural features of gram-negative bacteria?