4_Microbial Culture of Clinical Samples: Throat Swab Culture

Clinical microbiology

is an important aspect in the accurate diagnosis of a disease

Streptococcus pyogenes

• is the causative agent of pharyngitis, skin and soft tissue infections, and nonsuppurative infections as it can colonize the oropharynx, skin and tissue surfaces.

• , which are also called group A Streptococcus or group A strep, cause acute pharyngitis known as strep throat.

Streptococcus agalactiae

colonizes the female genital tract that may cause genital infections in older adults, pregnant women that may lead to neonatal infections.

Streptococcus pneumoniae

also colonizes the oropharynx that may lead to diseases such as sinusitis, otitis media, meningitis and pneumonia.

Streptococci

• are gram-positive spherical bacteria that characteristically form pairs or chains during growth.

• are widely distributed in nature.

• Some are members of the normal human microbiota

• elaborate a variety of extracellular substances and enzymes.

• are a large and heterogeneous group of bacteria, and no one system suffices to classify them

Urinary tract infections

are mostly caused by gram-negative rods which originate in the large intestines, contaminate the urethra, ascend into the bladder, and may migrate to the kidney or prostate. Infections in the kidneys may also be routed from the bloodstream

Escherichia coli

are mostly involved in urinary tract infections, as attributed to various virulence factors such as the ability to produce adhesins that bind to the cells lining the urinary bladder and upper urinary tract; the ability to produce hemolysin HlyA that can lyse RBCs and other cells which can trigger inflammatory reactions.

hemolysin HlyA

Escherichia coli are mostly involved in urinary tract infections, as attributed to various virulence factors such as the ability to produce adhesins that bind to the cells lining the urinary bladder and upper urinary tract; the ability to produce ___that can lyse RBCs and other cells which can trigger inflammatory reactions.

urine specimen

In the case of possible kidney or urinary tract infection, a ___will be collected for bacterial analysis

sore throat

If the patient has a ___, the throat will be swabbed, and this will be submitted for analysis.

SUPPORTIVE MEDIA

contain nutrients that allow most non-fastidious organisms to grow at their normal rate. These media do not give one organism any growth advantage over another. The organism's own metabolism affects the progress of its growth.

SELECTIVE MEDIA

• are semisolid plating media prepared by adding dyes, antibiotics, or other chemical compounds to certain media.

• These substances selectively inhibit the growth of certain microorganisms and permit the growth of others.

• are used for fecal and sputum specimens, where many normally occurring bacteria could obscure the presence of the pathogenic material.

ENRICHMENT MEDIA

• permit one organism to grow rapidly while inhibiting the growth of other organisms.

• are especially useful for the isolation of Salmonella or Shigella from stool cultures, which contain several bacteria -- normal intestinal flora -- that are so numerous that they would obscure the growth of pathogens.

Salmonella or Shigella

Enrichment media are especially useful for the isolation of ____ from stool cultures

Subculture to a solid plating medium

____from the enrichment broth must be made to obtain isolated colonies for final identification.

DIFFERENTIAL MEDIA

• contain dyes, indicators or other constituents that give colonies of particular organisms distinctive and easily recognizable characteristics

• The final identification of an organism often involves isolation on a suitable culture medium and then the characteristic reaction or growth on a differential medium.

• Such a characteristic reaction constitutes a confirmatory test for the microorganism.

CAP, BAP

Examples of Enriched Media:

BAP, MSA, MAC, EMB, HE

(Hektoen enteric agar)

Examples of Differential Media:

MSA, MAC, EMB, HE, PEA, CNA, MTM

(Hektoen enteric agar, Phenylethyl alcohol agar, Colistin nalidix acid agar, Modified Thayer-Martin agar)

Examples of Selective Media:

throat swab culture

is a laboratory procedure that is used to find and identify microorganisms in the back of your mouth. This serves as a guide in appropriate therapy in infectious and inflammatory diseases of the throat.

Cultivation

is a technique of growing microbes in culture from the site of infection ( disease.

fastidious

“Picky" microbes that need complex, exceptional media in order to grow are referred to as

non-fastidious

Those that are more basic and grow easily are referred to as

Phenylethyl alcohol Agar (PEA)

This medium is used for the isolation of most Gram-positive cocci. Partly inhibits Gram-negative bacteria.

MacConkey

Crystal violet agar, such as ___: This medium is selective for most Gram-negative microbes. Crystal violet dye and bile salts inhibit most Gram-positive organisms.

Crystal violet dye and bile salts

inhibit most Gram-positive organisms.

MSA Agar

This medium, containing 7.5% NaCl, inhibits most organisms other than salt-loving microbes (halophilic) such as Staphylococcus.

Blood Agar Plate (BAP)

The 5% or 10% sheep's blood that is incorporated into this agar is designed to enrich the media for the purpose of cultivation of fastidious organisms such as Streptococcus spp. It also enables the display of hemolysis patterns of some microorganisms, especially staphylococci and streptococci.

Alpha hemolysis

This is incomplete lysis of red blood cells, resulting in a reduction of hemoglobin to methemoglobin, resulting in a greenish zone around the bacterial colonies. Streptococcus pneumoniae and viridans streptococci are examples.

Beta hemolysis

This is complete lysis of red blood cells, resulting in complete destruction and utilization of hemoglobin, resulting in a clear zone surrounding the colonies. It is produced by 2 types of beta hemolysins: streptolysin O (antigenic, oxygen-labile enzyme) and streptolysin S (a nonantigenic, oxygen-stable enzyme). The hemolysis is enhanced when the blood agar plates are both streaked and stabbed, which may show subsurface hemolysis by streptolysin O due to the reduced oxygen tension underneath the surface of the agar

Neisseria spp and Haemophilus spp

Fastidious organisms are those that require special nutrients in order to grow and thrive and be isolated in the microbiology lab. Examples include ____ because they require blood, hemoglobin, various vitamins, sugars, and amino acids in order to grow and thrive.

Gamma-hemolysis

nonhemolysis

TRYPTICASE SOY AGAR WITH 5% SHEEP'S BLOOD

This type of agar, referred to as TSA II agar, is used to cultivate fasidious microbes and for the observation of hemolytic reactions. It is a general purpose media that allows a vast array of microbes to grow.

Hemolysis

lysing of the red blood cells in the agar by extracellular enzymes produced by certain bacteria

CHOCOLATE AGAR WITH 10% HORSE'S BLOOD

• is one of the non-selective and enriched agars used to cultivate grow. It is used to isolate pathogens such as fastidious organisms that have special requirements and needs for special nutrients in order to Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catharralis, Haemophilus influenzae, Francisella tularensis.

• It contains 10% horse's blood in which the red blood cells have been lysed by slowly heating the agar to 80 degrees Celsius.

• contains Factor V (NAD) and Factor X (hemin), both of which are needed by H. influenzae in order to grow

• . With addition of a Bacitracin antibiotic disk, the media also becomes selective for the which shows resistance to the disk.

• The peptone base supplies proteins to the bacteria.

fermentation results in changes in the pH (<6.8)

Enteric bacteria that ferment lactose, the sugar in the medium, appear to pinken the agar because____ , which are indicated by the pH indicator neutral red.

ammonium

Non-lactose-fermenters utilize peptone in the agar instead of lactose, which produces ___as a metabolic byproduct, raising the pH, leading to the formation of transparent colonies on the agar

E. coli, Klebsiella spp, Enterobacter spp, Citrobacter spp (slow), Serratia spp (slow)

Examples of lactose + organisms:

E. coli, Klebsiella spp, Enterobacter spp, Citrobacter spp (slow), Serratia spp (slow) E. coli (some strains), Acinetobacter, Salmonella, Shigella, Proteus, Pseudomonas

Examples of lactose - organisms:

HEKTOEN (HEK) AGAR

• is both selective and differential. Its main purpose is isolation of pathogenic bacteria such as salicin, peptone, pH indicators yellow and bromthymol blue, H2S indicators, and inhibitors of Gram-positive bacteria.

• Green or blue-green growth means that the organism does not ferment lactose.

• Pink to orange growth means that the organism produces acid from lactose fermentation.

• If the growth has a black center, the organism produces H2S from sulfur reduction.

Green or blue-gree

In Hektoen agar, _____ growth means that the organism does not ferment lactose.

Pink to orange

In Hektoen agar,  ___growth means that the organism produces acid from lactose fermentation.

black center

In Hektoen agar,  ___ means that the organism produces H2S from sulfur reduction.

H2S from sulfur reduction

In Hektoen agar,  black center means that the organism produces ___

MACCONKEY AGAR WITH SORBITOL

• This agar is a variation of traditional MacConkey agar in that it contains the sugar sorbitol instead of lactose.

• It is selective for the highly pathogenic Escherichia coli O157:H7 serotype, a non-sorbitol-fermenter on SMAC agar.

• It utilizes the peptone in the agar instead of the sorbitol, producing clear colonies on the agar.

YERSINIA CIN AGAR/CEFSULODIN-IRGASAN-NOVOBIOCIN

This agar is selective for isolation of Yersinia spp, such as Y. enterocolitica, a causative agent of gastroenteritis. It is a peptone base with yeast extract, mannitol, and bile salts supplemented with cefsulodin, irgasan and novobiocin, along with a neutral red indicator and crystal violet indicators.

TCBS AGAR (THIOSULFATE CITRATE-BILE SUCROSE)

This agar is a highly selective media utilized to isolate Vibrio cholerae and Vibrio parahaemolyticus 

CAMPYLOBACTER AGAR/SKIRROW AGAR

This medium consists of a peptone and soy protein base lysed with horse's blood. Antibiotics such as vancomycin inhibit the growth of Gram-positive organisms. Polymixin B and trimethoprim inhibit most Gram-negative organisms.

Campy-blood agar

contains vancomycin, trimethoprim, polymixin B, amphotericin B, and cephalthin in a Brucella agar base with sheep's blood.

MANNITOL SALT AGAR

• This agar is a selective and differential medium used for the isolation of S. aureus.

• Yellow growth means that the organism produces acid from the fermentation of mannitol in the medium.

• Red or pink growth means that the organism does not ferment the mannitol in the medium.

Endo agar

• is used to detect fecal contamination of water or dairy products.

• It contains color indicators such as basic fuchsin and sodium sulfite.

EOSIN METHYLENE BLUE AGAR

is used to isolate fecal coliforms such as E. coli and other members of the Enterobacteriacea

dark purple complex with a metallic green sheen

When large amounts of acid are produced during the fermentation process, a beautiful _____is produced. This is an indicator of heavy lactose and/or sucrose fermentation typical of fecal coliforms like E. coli.

CNA AGAR WITH BLOOD (Columbia Colistin-Nalidixic Acid Agar)

• is a nutritionally rich agar containing 5% sheep's blood blood agar and 10 mg of colistin per liter and 15 mg of nalidixic acid per liter, which promotes the growth of Gram-positive organisms and prevents the growth of Gram-negative ones.

• Three peptone (protein) sources are included in the agar mix, and it is a type of supportive and selective and differential media used to help differentiate bacterial colonies based on their hemolysis patterns.

MARTIN-LEWIS AGAR

This agar contains antimicrobials that are selective for the growth of Neisseria spp

NEW YORK CITY (NYC) AGAR:

• is selective for Neisseria gonorrhoeae.

• It consists of a peptone base with cornstarch, supplemented with yeast dialysate, 3% hemoglobin, and horse plasma. Antibiotics include vancomycin, colistin, amphotericin B, and trimethoprim.

THAYER-MARTIN AGAR

This agar is a Mueller-Hinton based agar containing 5% sheep's blood plus antimicrobials that prevent growth other than Neisseria spp bacteria, including the pathogenic Neisseria gonorrhoeae and Neisseria meningitidis.

SSA AGAR Streptococcal Selective Agar

is selective for S. pyogenes (Group A strep) for the isolation of the pathogens which cause strep throat.

CDC ANA AGAR WITH PEA

This anaerobic agar contains blood, phenylethyl alcohol, and antibiotics which prevent the growth of Gram-negative organisms and promote the growth of Gram-positive ones, especially Gram-positive cocci.

Xylose Lysine and Desoxycholate (XLD) agar

is selective and differential for Shigella species and for Salmonella species

BRAIN-HEART INFUSION (BHI) AGAR

This all-purpose fungal media is used for the initial isolation of yeasts and molds and contains antibiotics such as gentamicin to prevent the growth of bacteria. It is rich in nutrients and is available as a broth or as an agar. It may or may not contain blood. Several key ingredients are infused together for optimal growth, and they include several animal tissue sources, peptone (protein), phosphate buffer, and dextrose (sugar), which gives the bacteria the energy they need.

hyaluronic acid capsule

streptococcal cell wall is encompassed by a ___that is also antiphagocytic and serves as an accessory virulence factor.

Streptolysin O (SLO)

• Oxygen labile

• will know through stabbing the BAP agar

• responsible for haemolysis in the deeper layers of blood agar where streptococci grow in the absence of oxygen.

• toxic for red blood cells, white blood cells, as well as myocardial cells in tissue cultures

Streptolysin S (SLS)

• Oxygen stable

• responsible for the haemolytic zone around the streptococcal colonies growing on the surface of blood agar in the presence of oxygen. It is nonantigenic because of its low molecular weight.

S. pneumoniae

• a.k.a “the pneumococcus”

• Appears as lancet-shaped diplococci

• obligate parasite of humans

• found in the nasopharynx of 5 to 40% healthy individuals

flattened-depressed centers (draughtsman appearance)

Streptococcus pneumoniae colonies on blood agar