Skip to main content

My Clinical Microbiology 1 Notes

CLINICAL MICROBIOLOGY 1

001 Introduction to Bacterial Identification

Contents

  • Bacterial Taxonomy
  • Steps in Diagnostic Isolation and Identification of Bacteria
  • Isolation and Identification Method ofs
    • Isolation and Culturing of Microorganism
    • Microscopic Examination
    • Biochemical Testing of Microorganism
    • Antimicrobial Sensitivity Testing (AST)

1. Introduction

Infectious diseases caused by different bacteria have a variety of clinical courses and consequences

Isolation and identification of bacteria aids treatment

Susceptibility testing of isolates (e.g: establishing Minimum Inhibitory Concentration, MIC) can help in selection of antibiotics for therapy

Recognizing that certain species (or strains) are being isolated atypically may suggest that a disease outbreak has occurred

2. Bacterial Taxonomy

  • Taxonomy is the practice and science of classification
  • Family is a group of related genera
  • Genus is a group of related species
  • Species is a group of related strains
  • Type means sets of strain within a species (e.g: biotypes, serotypes)
  • Strain is one line or a single isolate of a particular species
  • Reviewing naming convention of bacterial species: Streptococcus pyogenes (abbreviation: S. Pyogenes)

3. Steps in Diagnostic Isolation and Identification of bacteria

Step 1 Isolate and observe growth in pure culture

  • Samples of body fluids (blood, urine, CSF) are streaked on culture plates
  • Isolated colonies of bacteria which are visible to the naked eye appear after incubation for one to several days
  • Observe colonies for size, texture, color, hemolytic reactions

Step 2 Microscopic examination

  • Colonies are Gram stained and individual bacterial cells observed under the microscope

Step 3 Species identification

  • The bacteria are speciated using the isolated colonies. This often requires an additional 24 hours of growth. Generally using physiological tests. 

Step 4 Antibiotic susceptibility testing

  • A test which uses antibiotic-impregnated wafers to test whether particular bacteria are susceptible to specific antibiotics

4. Isolation and Culturing of Microorganism

Culturing the organisms in dishes or tubes containing the nutrients required for microbial growth

To identify a pathogen it is necessary to isolate the organism in pure growth from a single colony

A pure growth refers to a culture which contains only a single species of organism

 A mixed culture is one which contain more than one species

5. Microscopic Examination

The appearance of bacteria is observed under the microscope

  • Gram positive
  • Morphology (rod, coccus, spiral, pleomorphic etc)
  • Arrangement (single, chains, pairs etc)
  • Size

Besides the Gram stain, there are other less commonly employed stains (e.g: spore, capsule stains)

6. Morphology of Bacteria

Most bacteria are classified according to shape

  • Bacillus: rod shaped
  • Coccus: spherical
  • Spirillum: spiral

Some bacteria have quite different shapes

  • Coccobacilli: elongated coccal form
  • Filamentous: bacilli that occur in long threads
  • Vibrios: short, slightly curved rods
  • Fusiform: bacilli with tapered ends

7. Arrangement of Bacteria Cells

Bacteria sometimes occur in groups, rather than singly, and the single cell's shape influenced the cell arrangements that they form through binary fission

Bacilli divide along a single axis, so sometimes they are seen in pairs or chains. Since they only divide along one axis, you will not find them in clusters, such as those formed by staphylococcal bacteria

Cocci divide on one or more planes, producing cells in

  • Pairs (diplococci)
  • Chains (streptococci)
  • Packets (sarcinae)
  • Clusters (staphylococci)

8. Bacterial Colony Morphology

Different types of bacteria will sometimes produce colonies with a distinctive appearance. The characteristics of a colony (shape, size, color, etc) are termed the colony morphology.

  • Shapes: punctiform (under 1 mm diameter), round, filamentous, irregular
  • Forms: circular, filamentous
  • Elevation: turn the petri dish on end
  • Margin: smooth (entire), curled, wavy, lobate, filamentous
  • Surface: smooth, glistening, rough, dull (opposite of glistening), rugose (wrinkled), concentric, contoured
  • Opacity: transparent, opaque, translucent (almost clear but distorted vision, like looking frosted glass), iridescent (changing colors in reflected light)
  • Chromogenesis (pigmentation): white, buff, red, purple

9. Biochemical Testing of Microorganism

OF glucose

Used to identify the oxidative and fermentative abilities of microorganisms. Organism to be tested in stabbed into 2 tubes of the semi-soft Agar medium. One tube remains open to air and one tube is covered with oil. Tubes are incubated and results read by noting the color of the bromthymol blue indicator. If glucose is used, acid end-products are generated, the pH is lowered and the bromthymol blue indicator turns yellow.

Open tube turns yellow=oxidative

Bromthymol blue green turns yellow=fermenting glucose

Alcaligenes faecalis: glucose not metabolized (non saccharolytic)

Escherichia coli: fermentative and oxidative metabolism

Pseudomonas aeruginosa: oxidative metabolism

Phenol Red Fermentation Broth

Used to identify acid and gas production from a variety of sugars such as lactose, mannitol, sucrose. Broths are inoculated and incubated. The results are determined by the color of the phenol red indicator. If sugar is fermented, acid end-products are generally and the phenol red indicator turns yellow. 

Staphylococcus aureus: alkaline, non glucose and lactose fermenter

Escherichia coli: acid, glucose fermenter and gas bubbles in Durham tube

Shigella sonnei: acid, glucose fermenter and no gas bubbles in Durham tube

Pseudomonas aeruginosa: glucose fermenter but non lactose fermenter, gas bubbles in Durham tube

Simmons Citrate Agar

Used to identify organisms that can utilize citrate as their sole carbon source. A slant is inoculated and incubated. The result is determined by determining the color of the bromthymol blue indicator. Citrate is used and the pH of the medium rises to turn the tube a bright Prussian blue color. 

Enterobacter aerogenes, Salmonella enteritidis, Salmonella typhimurium, Proteus vulgaris: positive

Escherichia coli: negative

MRVP (Methyl Red-Vogues Proskauer)

Used to identify organisms that ferment to produce stable acid end-products or neutralized acids into acetoin

MR: 

Escherichia coli: red, positive

Klebsiella, Enterobacter aerogenes: yellow/orange, negative

VP:

Escherichia coli: negative

Klebsiella, Enterobacter aerogenes: pink, positive

Urease test

Urease is an enzyme produced by some microorganisms. It attacks the nitrogen and carbon bond in aminde compounds and forms alkaline end product ammonia. The urease test is particularly helpful in identifying Proteus from other species due to the rapid action of the enzyme by Proteus compared to other non lactose fermenting enterics. The pH indicator phenol red changes to a deep pink color in the present of the alkaline ammonia. 

Escherichia coli: negative

Proteus vulgaris: pink, positive

Urease positive bacteria include Helicobacter pylori, Klebsiella, Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus aureus, Haemophilus influenzae, Mycobacteria except MAC, Cryptococcus, Ureaplasma, Diptheroids 

Indole production test

Tryptophan is an essential amino acid that can undergo oxidation by the action of the bacterial enzyme tryptophanase. Not all bacteria possess this enzyme and so this test can be used as a biochemical differentiator. The SIM Agar which contains tryptophan is used to identify the presence of indole. Kovac's reagent is added to the top of an SIM Agar deep that has been inoculated and incubated with a test organism. The formation of a cherry red reagent ring is a positive result. 

Escherichia coli, Proteus vulgaris, Vibrio cholerae: positive

Klebsiella pneumoniae, Enterobacter sp, Proteus mirabillis, Staphylococcus aureus, Salmonella arizonae: negative

Hydrogen Sulfide test

Gaseous H2S may be produced in 2 ways by bacteria. SIM Agar may be used to detect this production of gas. SIM Agar contains peptone, sodium thiosulfate, and ferrous ammonium sulfate. When H2S is produced, a precipitation reaction will occur with the ferrous ammonium sulfate. An insoluble black precipitate is seen as a positive result. 

Escherichia coli, Staphylococcus aureus: negative

Proteus vulgaris: positive

Triple Sugar Iron

May also used to determine H2S production as well as carbohydrate fermentation. 

Pseudomonas, Acinetobacter, Moraxella: K/K, K/NC, non fermenter, uses peptone

Escherichia coli, Klebsiella, Enterobacter, Serratia: A/A, ferments glucose, lactose and sucrose, gas 

Proteus: A/A, H2S

Shigella, Morganella, Providencia: ferments glucose, uses peptone, K/A, no gas

Salmonella: ferments glucose, K/A, H2S, gas 

Phenylalanine Deaminase test

Used to identify bacteria with the ability to deaminate phenylalanine into phenylpyruvic acid. After incubation, the slant is covered with ferric chloride solution. The ferric chloride will react with any phenylpyruvic acid present and produce a dark green color. A yellow color is a negative result. 

Escherichia coli: negative

Providencia stuartii: positive

10. Antimicrobial Sensitivity Testing (AST) 


002 Gram Positive Cocci

Contents

  • Genus: Staphylococcus
    • Species and Pathogenicity
      • S. Aureus
      • S. Epidermidis
      • S. Saprophyticus
    • Laboratory Diagnosis
      • Microscopy
      • Culture
      • Biochemical Reaction
      • Antimicrobial Sensitivity

1. Introduction

More than 30 species of staphylococci have been recognized

Gram-positive, non-motile, non sporing cocci

Occurs singly, in pairs, and in irregular clusters

Size may be variable

Colonies are opaque and maybe white or cream

Occasionally yellow or orange

Optimum growth temperature 30~37°C

Facultative anaerobes and have a fermentative metabolism

Salt tolerant and highly resistant to drying

Some species are hemolytic

2. Staphylococcus natural habitat

Staphylococcus aureus and Staphylococcus epidermidis are normal flora of the skin

Staphylococcus aureus colonizes moist environments (nasal passages and axillae)

Other species of staphylococci are infrequent human commensals

Staphylococcus epidermidis is rarely pathogenic, Staphylococcus aureus is an opportunistic pathogen

3. Staphylococcus aureus

Staphylococcus aureus can cause many forms of infection 

Superficial skin lesions (boils, styles) and localized abscesses

Deep seated infections in debilitated persons from chronic illness, trauma, burns or immunosuppression

Staphylococcus aureus is the major cause of hospital acquired infection of surgical wounds

Food poisoning (release of enterotoxin in food)

Toxic shock syndrome (release of superantigen into blood)

Staphylococcal scalded skin syndrome

4. Enzymes and toxins produced by Staphylococcus aureus

Coagulase

Leucocidin

Lytic toxin

DNAse

Hyaluronidase

Lipase

Staphilokinase

Exfoliatin

Enterotoxin B 

Beta lactamase

5. Other species

Staphylococcus epidermidis causes infections associated with inwelling medical devices include artificial valve endocarditis, catheter and shunt infections, and prosthetic infections

Staphylococcus saprophyticus is mainly associated with cystitis, pyelonephritis and acute urethritis in females

Laboratory Diagnosis

6. Identification

7. Microscopy

Direct gram stain of specimen collected from the lesion/wound/pus/sputum

Blood samples require culturing first because of the small numbers of bacteria in blood

8. Culture

Primary isolation media

Material from clinical specimen or blood culture streaked onto media

Blood Agar: 16~48 h incubation in 5~10% CO2 at 35~37°C

Specimens likely to be contaminated with other microbes plated on mannitol salt Agar

Colonial morphology

Varies with species

Usually opaque and maybe white or cream

Sometimes yellow to orange on Blood Agar

Hemolysis may be detected

MSA is both selective and differential

It contains 7.5% NaCl which selects for organisms that are halotolerant

It also contains mannitol and phenol red

Organisms able to ferment mannitol produce acid fermentation products which lowers pH

Phenol red indicator turns yellow

Staphylococcus aureus is able to ferment mannitol

Other staphs grow on MSA but do not ferment mannitol

9. Biochemical Reaction

Catalase test

Catalase is an enzyme produced by most aerobic and facultative anaerobe bacteria. It decomposes hydrogen peroxide into oxygen and water. Some bacteria from hydrogen peroxide as an oxidative end product of aerobic breakdown of sugars. If allowed to accumulate is highly toxic to bacteria. Enzyme present in staphylococci but lacking in streptococci. Bubbling indicates a positive result. Scant or no bubbling indicates a negative result. Catalase test should not be done on Blood Agar (blood itself will produce bubbles). 

Coagulase test

Coagulase is an extracellular protein which through the conversion of fibrinogen to fibrin results in the clotting of blood plasma. It exists in 2 forms: bound coagulase (clumping factors) that bound to cell wall and free coagulase that liberated by the cell wall. It present in Staphylococcus aureus but lacking in most other staphylococci. Some isolates of Staphylococcus aureus may be defective in coagulase and give a negative result. 

DNAse test (Staphylococcus aureus is DNAse positive)

Modified oxidase test to differentiate micrococci from staph

Lysostaphin test (commercial identification kit)

10. Antimicrobial Sensitivity

Antibiotic against Staphylococcus aureus is penicillin, cephalosporin, erythromycin, lincomycin, Clindamycin, vancomycin

Many strain of Staphylococcus aureus are penicillin-resistant due to production of penicillinase (a beta lactamase)

Novobiocin is a antimicrobial drug

Mode of action is to prevent supercoiling of DNA by DNA gyrase (Inhibitory effect on DNA replication)

Most strains of Staphylococcus saprophyticus are resistant whereas most strains of Staphylococcus epidermidis are sensitive (zone of inhibition >17 mm)


003 Gram Positive Cocci

Contents

  • Genus: Streptococci
    • Species and Pathogenicity
    • Streptococcus pyogenes (Lancefield Group A)
    • Streptococcus agalactiae (Lancefield Group B)
    • Streptococcus enterococci (Lancefield Group D)
    • Streptococcus pneumoniae
  • Laboratory Diagnosis
    • Microscopy
    • Culture
    • Biochemical Reaction
    • Antimicrobial Sensitivity

1. Introduction

Large group of Gram positive bacteria

Inhabit a wide range of hosts as normal microbial flora including humans, horses, pigs and cows

Can cause diseases from subacute to acute or chronic

Taxonomic note: 

Streptococci and related organisms have undergone extensive taxonomic revisions

Phenotypic methods alone may not adequately identify organisms to species level

Further identification (molecular methods) may be necessary where indicated clinically or epidemiologically

2. General characteristics

Gram-positive cocci often occuring in pairs and chains

On Blood Agar, species exhibit various degrees of hemolysis

2 important factors in presumptive identification: hemolytic activity of colonies on Blood Agar and Lancefield serological grouping

Facultatively anaerobic

Catalase negative

Metabolizes carbohydrates fermentatively (major metabolite produced is lactic acid)

3. Lancefield classification

Based on proteins found in cell walls of streptococci

Species classified into groups A through Medically important are Group A, B and D

4. Species of medical importance

Streptococcus pyogenes (Group A)

Streptococcus agalactiae ( Group B)

Enterococcus sp (Group D)

Streptococcus pneumoniae (do not possess Lancefield protein)

Viridans streptococci (Alpha hemolytic streptococci that lack Lancefield protein)

More than 40 species of streptococci and related organisms have been reported to cause human infection

5. Streptococcus pyogenes (Group A)

Pharyngitis and cellulitis

Impetigo, Streptococcal toxic shock syndrome

Rheumatic fever (inflammation of body's organ system)

Glomerulanephritis 

Enzymes and toxins produced by Streptococcus pyogenes

Streoptolysin o

Streptolysin S

Streptokinase

Hyaluronidase

DNAse

DPNase

Erythrogenic toxin

6. Streptococcus agalactiae (Group B)

Causes neonatal meningitis and septicemia

After transmission from normal vaginal flora of mother

7. Enterococcus

Found in the gut flora 

Infection often follows from fecal contamination

Significant cause of UTI

Opportunistic infection (intraabdominal, septicemia and endocarditis)

8. Streptococcus pneumoniae

Leading cause of pneumonia in all ages often follows viral infection

Causes otitis media

Often spreads causing bacteremia and meningitis

9. Identification

10. Microscopy

Gram stain

Streptococcus and Enterococcus are Gram positive, round or ovoid cells occuring in pairs, short or long chains 

Streptococcus pneumoniae is Gram-positive, lanceolate cells occuring in pairs, often with a visible capsule

Non-motile 

Some may have capsule

11. Culture

Hemolysis on Blood Agar

Types of hemolytic activity demonstrated by streptococci: 

Alpha hemolysis

Partial lysis of the RBCs surrounding a colony, causes a greenish discoloration of the medium

Beta hemolysis 

Complete lysis of RBCs surrounding a colony, causes a clearing of the blood from the medium

Non-hemolytic (Gamma hemolysis)

No hemolysis takes place

No change in the media surrounding a colony

Primary isolation media

Blood Agar

Incubated at 5~10% CO2 at 35~37°C for 16~48 h

Throat swabs: anaerobically at 35~37°C for 16~48 h

Staph/Strep Agar

In air at 35~37°C for 16~48 h

CLED Agar

In air at 35~37°C for 16~48 h

Colonial appearance


12. Biochemical Reactions

Catalase test

As described with Staphylococcus, streptococci and morphologically similar organisms are usually catalase negative

Bile Aesculin Hydrolysis test used to differentiate Enterococci from Streptococci. Ability of the bacterium to grow in the presence of 40% bile and produce aesculinase. The aesculetin combines with ferric ions in the medium to form a black complex. 2 methods of examining results: examine for presence of dark brown to black halo around bacterial growth (negative has no color change) and examine under UV light at 360 nm (positive has no fluorescent; negative has fluorescent). Aesculin is a glycoside composed of glucose and a dihydroxycoumarin compound. Certain bacteria such as the Enterococcus are able to grow in the presence of bile and hydrolyze the esculin. The liberated dihydroxycoumarin complexes with ferric citrate present in the media to form a dark brown/black soluble compound. Enterococci, Lancefield Group D Streptococci hydrolyze esculin in 40% bile while others don't. 

Streptococci Grouping (commercial kit)

PYR uses L-pyrrolidonyl-Beta-naphthylamide to detect the presence of an enzyme (L-pyrrolidonylarylamidase). Product of hydrolysis reacts with a reagent (N, N-methyl-aminocinnamaldehyde) to produce a red color. Enterococci and Streptococcus pyogenes are positive. A small amount of colony is rubbed on the surface of a PYR disc (available commercially). A drop of detector reagent is added. Observed for red color within 5 minutes. (Negative is orange/yellow color, no color change). 

Bile solubility (optional) is used to differentiate between Streptococcus pneumoniae and other Alpha hemolytic streptococci. It determines ability of bacterial cells to lyse in presence of bile salts within a specific time. Streptococcus pneumoniae possess an autolytic enzyme which lyses the cell's own wall during division. Addition of bile salts (sodium deoxycholate) activate the autolytic enzyme and the organism rapidly autolyse. Other Alpha hemolytic streptococci do not possess this system and do not dissolve in bile. Rapid lysis of the pneumococcus in the presence bile cause by the activation of cell wall autolytic enzyme as a consequence of the detergent action of bile. 

CAMP test (Christie, Atkins, Munch, Peterson) to identify Group B Streptococci. The hemolytic activity of streptococcal-hemolysin on RBC is enhanced by an extracellular factor produced by Group B Streptococci called the CAMP factor. Therefore, wherever the 2 reactants overlap in sheep BAP, an accentuation of the Beta-hemolytic reaction will be noted. 

Single streak of the streptococcus perpendicular to a strain of Staphylococcus aureus that is known to produce beta hemolysin is made. The 2 streak lines must not touch one another. Plate incubated with an ambient atmosphere (room temperature). The plate should not be incubated in an anaerobic environment because many Group A Streptococci are positive in the absence of oxygen. Group B Streptococcus are positive. 

13. Antimicrobial Sensitivity Testing (AST)

Optochin Sensitivity Test detects the organisms susceptibility to the chemical optochin. The chemical tests fragility of the bacterial cell membrane. It differentiates Streptococcus pneumoniae from other alpha hemolytic strep. Optochin disc placed at edge of primary inoculum on blood plate of specimen. Incubate as for Blood Agar culture. Positive result: zone of inhibition of ≥5 mm radius from edge of disc. Organism is Streptococcus pneumoniae. Negative result: no zone or zone <5 mm radius. Organism is not Streptococcus pneumoniae. Optochin causes Streptococcus pneumoniae to lyse due to changes in surface tension. 

Bacitracin Sensitivity Testing differentiates beta hemolytic group A Streptococci from other beta hemolytic streptococci. Bacitracin is an antibiotic produced by Bacillus subtilis. It is used as topical antibiotic. It inhibits cell wall synthesis. Growth of Group A Streptococcus pyogenes is inhibited by low concentrations of bacitracin on Blood Agar. Most other staphylococci are not inhibited. Diameter of zone of inhibition (>10 mm = sensitive). Streptococcus pyogenes is sensitive to bacitracin and will not grow around the antibiotic disc. Other beta hemolytic streptococci are not sensitive to bacitracin. 


004 Gram Negative Rods

Contents

  • Species and Pathogenicity
    • E. Coli
    • Enterobacter
    • Proteus spp. 
    • Klebsiella spp. 
    • Serratia
  • Laboratory Diagnosis
    • Microscopy
    • Culture
    • Biochemical Reaction
    • Antimicrobial Sensitivity

1. Species and Pathogenicity 

Enterobactericae is a large family of Gram negative rod. It is found in colon as part of normal flora. It can cause variety of diseases with different pathogenic mechanisms. 


2. General Features 

The family enterobactericae is facultative anaerobes. 

All ferment glucose. 

None have cytochrome oxidase. 

All contain endotoxin in their cell walls. 

Non spore forming. 

All members are similar in morphology and cultural characters. 

All can be differentiated by biochemical reaction. 

Gram-negative bacilli (rods)

Mobile with peritrichate flagella

Some are non motile (Klebsiella, Shigella)

3. Escherichia Coli

E. Coli is the majority of GI normal flora (T/F). It is one of the opportunistic pathogens. It can cause extraintestinal infection include UTI, septicemia and neonatal meningitis. 

Extraintestinal E.Coli infections are the common causes of septicemia and meningitis in neonates. 40% of gram negative meningitis cases is caused by E. Coli. 

Newborn acquires infection in birth canal just before or during delivery when mother's vagina is heavily colonized. 

4. Klebsiella

Gram-negative bacilli, short, non-motile, capsulate

Commensal in intestine

Klebsiella pneumoniae causes Fried-Lander pneumonia (severe fatal bronchopneumonia), neonatal meningitis, lung abscess, wound infection and UTI. 

5, Enterobacter

Motile

Colony morphology resembles Klebsiella on MacConkey

Most commonly isolated species include E. Cloacae, E. Aerogenes. Both have been isolated from wounds, urine, blood, and CSF. 

6. Serratia

Opportunistic pathogens associated with nosocomial outbreaks 

Ferments lactose slowly

Resistant to multiple antibiotics

S. Marcescens 

  • Species that is clinically important
  • Produces characteristic pink to red pigment (prodigiosin)
  • Cause hospital acquired infections of urinary or respiratory tract
  • Bacteremic outbreaks in nurseries and cardiac surgery and burn units

7. Proteus

Normal intestinal flora

Opportunistic pathogens

Cannot ferment lactose

4 species

2 species human pathogens include P. Mirabilis and P. Vulgaris. Both have been isolated from urine, wounds and ear and bacterimic infections. 

8. Laboratory Diagnosis: E. Coli Diarrhea

Specimen: stool

Microscopic examination of fresh preparation

Isolation on MacConkey Agar plates

Identification by sugar fermentation

9. Laboratory Diagnosis: Klebsiella

Specimen: sputum, urine, pus

Microscopy: Gram stain shows short Gram-negative rods with unstained halo around them due to the presence of the capsule

Isolation: on Blood Agar and MacConkey Agar media, it shows mucoid lactose fermenting colonies

10. Laboratory Diagnosis: Proteus

Specimen: urine, pus

Microscopy: Gram-negative rods

Culture: 

swarming growth occurs on Blood Agar

Non-lactose fermenting colonies occurs on MacConkey Agar

Identification: sugar fermentation (rapid urease activity)

11. Double Sugar Iron Agar Reaction

Differential Agar slants contain more than one sugar, and possess other indicators. In these slants, both respiration (aerobic) and fermentation (anaerobic) growth can take place. It is a very useful media for distinguishing among this group of organisms. 

This media contains lactose and glucose, in a ratio of 10 lactose molecules to 1 glucose molecule, a ferric salt, and phenol red as indicator (yellow below pH 7, red above). 

It is inoculated by stabbing into the butt of the tube with a needle, withdrawing the needle and streaking the surface of the slant. 

Slant color:

Red does not ferment either lactose or sucrose

Yellow ferments lactose and/or sucrose

Butt color:

Red does not ferment glucose

Yellow some fermentation of glucose has occurred, acid has been produced

Gas formed seen as cracks in the Agar, bubbles or the entire slant may be pushed out of the tube

Black precipitate means H2S has been produced

Results: 

E. Coli

  • Facultative anaerobe
  • Ferments glucose and lactose as a carbon source
  • Produce gas
  • Does not produce H2S

Klebsiella pneumoniae

  • Facultative anaerobe
  • Ferments glucose and lactose as a carbon source
  • Produces gas
  • Does not produce H2S

Enterobacter aerogenes

  • Facultative anaerobe
  • Ferments glucose and lactose as a carbon source
  • Produces gas
  • Does not produce H2S

Proteus vulgaris

  • Ferment glucose but not lactose as carbon source
  • Produces H2S

12. Enterotube Reactions

Yet, another set of media to differentiate among these genera is the commercially available Enterotube II (Roche Diagnostics, Montclair, N.J), which contains biochemical tests in miniature form.

Note: the percentage shown above a test in the images below indicates the likelihood (as percent) that a particular colony /clone of that specific organism (genus, species) would yield a positive test.


005 Gram Negative Rods

Contents

  • Species and Pathogenicity
    • Salmonella spp. 
    • Shigella spp.
  • Laboratory Diagnosis
    • Microscopy
    • Culture
    • Biochemical Reaction
    • Antimicrobial Sensitivity

1. Species and Pathogenicity

a) Salmonella

Gram-negative, facultative anaerobic rods

Clear, colorless colonies

Non-lactose fermenters

Most produce H2S

2 species include S. Enterica and S. Bongori

Antigenic structures

  • O antigen
    • LPS on outer membrane 
    • heat stable
  • Flagellar H antigen
  • Vi antigen capsular antigen
    • Surface polysaccharide 
    • prevents phagocytosis
    • found in serotype Typhi (used in identification)

Humans are infected by ingesting organisms in food, water and milk contaminated with human or animal excreta. 

Salmonella ser. Typhi and ser. Paratyphi have no known animal reservoir. Infections only occur in human. 

Other Salmonella infect various animals become reservoirs and sources for human infection. 

Enterocolitis: S. Enteritidis, S. Typhimurium, S. Choleraesuis 

• Defined as an infection in the lining of the small intestine.

• It occurs when you swallow food or water that is contaminated with the Salmonella bacteria.

– Source primarily poultry, milk, eggs, egg products

• Insufficiently cooked

– Handling infected pets

• Transmission

– Cooking utensils to prepare contaminated meat can spread contamination to other food (e.g. chicken to salad)

– Direct person-to-person reported in institutions.

• Dehydration from diarrhea, especially in young children and infants, is a dangerous complication

Septicemia: S. Choleraesuis, S. Typhimurium, S. Enteritidis, S. Hirschfeldii

• Accounts only about 5-10 % of Salmonella infections

• Occurs in one of two settings

– A patient with chronic disease such as sickle cell anemia / cancer. Example of type of infection is osteomyelitis in a child with sickle cell anemia.

– Child with enterocolitis

Enteric fever: S. Typhi, S. Paratyphi A, S. Schottmuelleri, S. Hirschfeldii 

• Typhoid fever

• Caused by Salmonella ser. Typhi

• Infection begins in small intestine, multiply

• Originate from infected individuals or carriers

• No known animal reservo

• Occurs most often in tropic & subtropic countries

• Improper sewage disposal, poor sanitation, lack of modern water systems assoc with outbreaks when organism reaches water source

• Carriers (esp. food handlers) are important source of infection

– Paratyphoid fever

• Similar to typhoid fever but less severe & fatality rate lower

b) Shigella

Not normal gastrointestinal flora

All species cause biliary dysentery (bloody diarrhea)

Presence in blood, mucus and pus in stool

Non-motile

Generally form clear, non-lactose fermenting colonies

Not produce H2S

Shigella are fragile organisms

Susceptible to physical and chemical agents (disinfectants, high concentration of acids or bile)

4 biochemically similar species

Divided into 4 major O antigen groups

  • Shigella dysentery (group A)
  • Shigella flexneri (group B)
  • Shigella boydii (group C)
  • Shigella sonnei (group D)

All have O antigen

Some strains have K antigen

Clinical infections

All species cause dysentery

Vary in epidemiology, mortality, severity of disease

Most common in developing countries

Shigella dysenteriae is the most virulent species with significant morbidity and mortality

Shigella boydii

Baciliary dysentery (enterocolitis)

Penetration of intestinal epithelial cells

Attachment of organism on mucosa

Local inflammation

Shedding of intestinal lining

Formation of ulcers following epithelial penetration

Symptoms vary from asymptomatic to severe

High fever, chills, abdominal cramps

Pain

Symptoms appear 24~48 hrs after ingestion of organism

Multiple in small intestine and move to colon

Watery diarrhea

Followed by bloody stools containing mucus and leukocytes

Patients suffer from extremely painful bowel movements

Some cases are life-threatening especially if extra intestinal complications develop

2. Specimen collection and transport

In enterocolitis, the organism is most likely easily isolated from a stool sample

In enteric fever, a blood culture is done for 2 weeks of illness

Bone marrow cultures are often positive

Sputum from hospitalized patients containing enteric bacteria need complete identification

3. Culture

MacConkey

  • 16~24 hrs incubation in air 35~37°C
  • Pink (lactose fermenting) or colorless (non-lactose fermenting)
  • Size and shape vary

CLED

  • 16~24 hrs incubation in air 35~37°C
  • Yellow (lactose fermenting) or blue (non-lactose fermenting)

Xylose-Lysine-Deoxycholate Agar (XLD)

  • Stool sample or black halo

4. Identification

Conventional biochemical tests

Commercial identification systems

5. Growth on MacConkey Agar

6. Double Sugar Iron Agar Reaction

Differential agar slants contain more than one sugar, and possess other indicators.

In these slants both respiration (aerobic) and fermentation (anerobic) growth can take place.

It is a very useful media for distinguishing among this group of organisms.

Double Sugar Iron Agar slant contains lactose and glucose, in a ratio of 10 lactose molecules to 1 glucose molecule, a ferric salt, and phenol red as indicator (yellow below pH7, red above).

It is inoculated by stabbing into the butt of the tube with a needle, withdrawing the needle and streaking the surface of the slant.

Results: 

Salmonella enteritidis ser Typhimurium

  • Ferments glucose but not lactose as a carbon source
  • Produces gas and H2S

Salmonella Typhi

  • Ferments glucose but not lactose as carbon source
  • Produces H2S
  • Unlike S. Enteritidis ser Typhimurium, does not produce gas

Shigella boydii

  • Ferments glucose but not lactose as carbon source
  • Does not produce gas or H2S

7. Enterotube Reactions

Yet, another set of media to differentiate among these genera is the commercially available Enterotube II (Roche Diagnostics, Montclair, N.J), which contains biochemical tests in miniature form.

Note: the percentage shown above a test in the images below indicates the likelihood (as percent) that a particular colony /clone of that specific organism (genus, species) would yield a positive test.


006 Gram Negative Rods

Genus

  • Species and Pathogenicity
    • Campylobacter
    • Helicobacter
    • Vibrio
  • Laboratory Diagnosis
    • Microscopy
    • Culture
    • Biochemical Reaction
    • Antimicrobial Sensitivity

1. Species and Pathogenicity

a) Campylobacter: Campylobacter jejuni and Campylobacter coli

Important properties:

Gram-negative bacilli

Actively motile

Small, slender, spirally shaped

Microaerophilic (5%)

Grow best at 42~43°C

b) Helicobacter

Morphology and characteristic:

Gram negative

Spiral

Biochemically inactive

Motile, unipolar

Noncapsulated

Produces a very powerful urease, catalase and oxidase

Strictly microaerophilic

c) Vibrio

Found as natural resident of aquatic environments

Most important pathogens of human are

  • Vibrio cholera
  • Vibrio parahaemolyticus
  • Vibrio vulnificus

Morphology:

Gram negative

Single polar flagellum

Curved or rod shaped

Noncapsulated

Non sporing

2. Laboratory Diagnosis: Microscopy, Culture, Biochemical reaction, Antimicrobial sensitivity

a) Campylobacter

Laboratory diagnosis:

  • Diarrhea-stool sample culture on Skirrow's medium (blood agar containing antibiotics inhibit other fecal flora) 
  • Plate incubate at 42°C, in microaerophilic (5%)
  • Identified by failure to grow at 25°C
  • Oxidase positive

Microscopy: 

pleomorphic (many kinds of shapes)

Close up view of Campylobacter jejuni on Blood Agar illustrating raised, gray-white and somewhat mucoid colonies

Gram stain of Campylobacter jejuni illustrating pleomorphic gram-negative bacilli, some short and curved, others forming spirals

b) Helicobacter

Direct microscopy biopsies

  • Warthin-starry silver staining
  • Haematoxylin-eosin staining
  • Gram staining: short curved, spiral gram negative bacteria

Culture

  • Inoculated on chocolate agar and campylobacter agar (Skirrow's)

Non-cultural diagnosis

Biopsy urease test

  • Biopsy tissue in urea solution containing a pH indicator
  • Positive within 2 min to 2 hrs if H. pylorus is present

Urea breath test

  • Urea C¹³ or C ¹⁴
  • If positive the isotope gives high reading in the CO2 emitted in the breath

c) Vibrio

Culture

Specimen: watery stool, rectal swab

Nutrient Agar: moist, round disc colonies

MacConkey Agar: colorless then pink

Blood Agar: do not produce hemolysis

TCBS media: V. Cholerae produces yellow colonies (sucrose), V. Parahaemolyticus and V. Vulnificus produce green colonies (non-sucrose fermenting)

Transport or holding media

  • Venkataraman-Ramakrishnan (VR) medium
    • Contains 20gm salt and 5g peptone in 1L dH2O
    • Vibrios not multiply, remain viable for several weeks
  • Cary-Blair medium
    • Suitable transport medium for Salmonella, Shigella and Vibrio
  • Enrichment medium
    • Alkaline peptone water

Biochemical reaction

  • Ferment glucose, mannitol, sucrose, maltose with acid production
  • Non-lactose fermenting
  • Catalase and oxidase positive

Other tests: growth of V. Parahaemolyticus in 8% NaCl


007 Gram Negative Cocci and Rods

1. Gram Negative Cocci

Medically relevant organisms

Neisseria gonorrhoeae: Gonorrhoeae

Neisseria meningitidis: Meningitis

Moraxella catarrhalis: Respiratory illness

Acinetobacter baumanii

Coccobacilli (rod in exponential phase, spherical in stationary phase)

Associated with nosocomial infections

Bacteremia, secondary meningitis, ventilator associated pneumonia

2. Lab Diagnostic Identification

 Most Neisseria, Moraxella and Acinetobacter can grow on simple media, under varying temperatures

N. Meningitidis and N. Gonorrhoeae (fresh isolates) are sensitive to fatty acids and trace metals present in media 

Require an enriched medium containing blood or blood products

CO2 atm and the incubation at 37°C required for growth

3. Culture


4. Microscopic Appearance

Neisseria gonorrhoeae

Gonococcus

Diplococci 

Coffee-bean shape

Fragile, do not survive long outside human body

5. Identification Scheme for Gm Neg Aerobic Cocci

Positive Oxidase Test

Dip a sterile cotton-tipped wooden stick into a fresh dye solution

Press the cotton tip against the colony to be tested

A colour change within 10 seconds indicated a positive result

Detects presence of cytochrome oxidase (enzyme) using a dye 

The dye is colorless in the reduced state. Purple in oxidized state.

Oxidase Test

Positive test commonly used for presumptive identification of Neisseria sp. 

Organisms contain large amounts of the enzyme (cyt. oxidase)

M. Catarrhalis and some Gram negative bacilli (Pseudomonas) also give positive test

Ability to grow on NYC media

NYC media is an enriched media containing antibiotics (Vancomycin, colistin, nystatin and trimethoprim)

NYC media permits the growth of the pathogenic N. Meningitidis and N. Gonorrhoeae but not the nonpathogenic Neisseria, Moraxella catarrhalis and other gram negative organisms

Carbohydrate degradation

Oxidative breakdown of the sugars glucose, maltose and sucrose can help to distinguish among Neisseria meningitidis, Neisseria gonorrhoeae and Moraxella catarrhalis


Haemophilus 

6. Important properties

Catalase positive, oxidase positive, and ferments glucose

Small, gram-negative rod

Growth factors 

7. Morphology

Pleomorphic Gram-negative rods

In clinical specimens, haemophilus is small, uniform coccobacillus

In cultures, haemophilus is longer, filamentous rods

8. Growth factors

All require blood for growth

Requirement based on X and V factors

X factor is haemin

V factor is nicotinamide adenine dinucleotide (NAD) or phosphate (NADP)

9. Growth on laboratory media

Chocolate Agar with X and V factors

Horse or Rabbit Blood Agar with X and V factors

Ordinary Blood Agar contains low level of V factor

S. Aureus excrete V factor

Growth around S. Aureus colonies is called satellitism

10. Diseases

Meningitis

Epiglottis

Bacteremia

Septic arthritis

Pneumonia

Otitis media, sinusitis

Cellulitis 

Occur in children 6 months to 6 years

Polysaccharide capsule is the major virulence factor

11. Specimens

CSF

Blood

Throat swab

Sputum, pus

Collected in sterile containers, should never be refrigerated

12. Culture

CSF is plated promptly on Blood Agar or Chocolate Agar

Blood culture is usually positive in case of epiglottitis and pneumonia

Sputum culture is homogenized by shaking with sterile water and glass beads for 15~30 mins

H. Influenzae requires X and V factors

Grows on Chocolate Agar

Satellitism around S. Aureus on Blood Agar

13. Satellitism

H. Influenzae streak on Blood Agar

S. Aureus streak across the same Blood Agar plate

Incubate

The colonies of H. Influenzae will be large alongside of S. Aureus

X factor is available in high concentration near streptococci growth

Identified by its colony morphology, satellitism, Gram-staining

14. X and V requirement of Haemophilus sp


15. Direct microscopy

Colonies of H. Influenzae appear as convex, smooth, pale, gray or transparent colonies

Gram-negative, coccobacilli, with no specific arrangement

Catalase and oxidase positive

16. Important properties of Brucella

Small, Gram-negative rods

Without capsule

3 animal reservoirs are B. Melitensis (goats and sheeps), B. Abortus (cattle), B. Suis (pigs)

Brucellosis is also considered an occupational disease because of a higher incidence in people working with animals (slaughterhouse cases)

17. Laboratory Diagnosis

Requirements of CO2 for growth

Production of urease and H2S

Use of specific antiserum

Grow slowly on Blood and Chocolate Agar

Fail to grow on MacConkey, EMB or other enteric media

Brucella sp are not included in the databases of commercially available kits for the identification of gram-negative organisms

18. Moraxella

Gram-negative, aerobic, oxidase positive

Diplococcus

Rarely caused infections in humans


008 Gram Positive Bacilli

Contents

  • Species and Pathogenicity 
    • Bacillus
    • Cornyebacterium
    • Listeria
  • Laboratory Diagnosis
    • Microscopy
    • Culture
    • Biochemical Reaction
    • Antimicrobial Sensitivity

Species and Pathogenicity

1. Bacillus anthracis

Disease

Cause anthrax, common in animal, rare in human

Human disease occur in 3 main form: cutaneous, pulmonary and gastrointestinal

2001 outbreak 18 cases, 5 died

2. Important properties

Large Gram-positive rod with square end 

Found in chains

Non motile

Its antiphagocytic capsule is composed of D-glutamate

3. Transmission

Spores of organism persist in soil for years

Cutaneous: Human most often infected (trauma to the skin). Allow the spores on animal products (wool, bristles) enter the skin

Gastrointestinal: Contaminated milk are ingested

4. Clinical findings

Cutaneous anthrax

95% of human cases of anthrax

Route of entry: skin

Sites involved: face, neck, hands, arms and back

Malignant Pustules: satellite lesions filled with serum or yellow fluid arranged around a central necrotic lesion which is covered by a black eschar.

Gastrointestinal anthrax: vomiting, abdominal pain and bloody diarrhea

Pulmonary anthrax: non specific respiratory tract symptoms (influenza)

Dry cough and substernal pressure

Death

5. Lab Diagnosis

Microscopy morphology

Gram stain, large square ended Gran positive rods, may appear end to end giving a bamboo appearance

Colonical morphology

Non hemolytic on 5% Blood Agar, raised large, grayish white 

6. Cornyebacterium sp. 

Found as free living saprophyte

Members of usual flora of humans and animals

Cornyebacterium diphtheriae is the most significant pathogen 

Other species may cause infections in immunocompromised host 

7. Important properties

Gram-positive rods 

Has a characteristic"Chinese letter" arrangement, often with club-shaped swellings at the poles with irregularly staining segments or granules

8. Transmission

Human is the only natural host

Toxigenic and non toxigenic organism reside in the upper respiratory tract

Transmitted by airborne droplet

Also infect skin at the site of preexisting skin lesion

Occur in tropic

9. Pathogenesis

Organism establish and maintain itself in the throat

Exotoxin production

  • Diphtheria toxin
  • Inhibit protein synthesis
  • Affect all eukaryotic cell

10. Clinical findings

Thick gray, adherent pseudo membrane over the tonsils and throat

Fever, sore throat, malaise

Toxin absorbed to bloodstream carried systemically

11. Growth of diphtheria bacilli


12. Identification

Microscopy

Gram stain: Gram-positive bacilli, Chinese letter pattern

Albert's stain for metachromatic granules

13. Culture 

Isolation of bacilli requires media enriched with blood, serum or egg

Blood Agar

Loeffler's serum slope

Rapid growth, 6~8 hrs 

Tellurite Blood Agar

Tellurite is reduced to tellurium, gives gray or black color to the colonies

14. Listeria monocytogens

General characteristics

Gram positive

Widespread in nature

Known to infect a wide variety of animals

Able to grows well at cold temperatures

15. Disease

Immuno suppressed adults

  • Febrile gastroenteritis
  • Meningitis and sepsis
  • Renal transplant patients

Newborns

  • Meningitis and sepsis
  • Transmission across the placenta or during delivery

16. Clinical findings

Newborn is infected at the time delivery can have acute meningitis 1~4 weeks later

The infected mother either is asymptomatic or has an influenza like illness

Watery diarrhea, fever, headache, abdominal cramps

Little vomiting

17. Laboratory Diagnosis

Gram staining

  • Appearance of gram positive rods
  • Resembling diphteroids 

Culture

  • Small, gray colonies with narrow zone of beta hemolysis on Agar plate
  • Confirmed with the presence of motile organism 


009 Mycobacterias and Spirochetes

Contents

  • Species and Pathogenicity
    • Mycobacterium
    • Sphirochete
  • Laboratory Diagnosis
    • Microscopy
    • Culture
    • Biochemical Reaction
    • Antimicrobial Sensitivity

1. Mycobacterias

Fungus-like bacteria, Acid-fast bacteria

Slender bacilli

Show branching filamentous forms resembling fungal mycelium

Difficult to stain, but resist decolorization

Aerobic, non motile, non capsulated, non sporing

2. Mycobacterium tuberculosis (Koch's bacteria)

• Important properties

Grows slowly (doubling time 18 h)

Clinical specimen must be held for 6~8 weeks before being record as negative

Obligate aerobe 

Resistant to acids and alkalis (NaOH concentrate specimens)

Resistant to dehydration

• Disease

Tuberculosis

Cause more death than any other single microbial agent

Humans are the natural reservoir

Transmitted from person to person via aerial route

The droplet are expelled during coughing, sneezing and even talking

• Laboratory Diagnosis

Sputum is collected in clean wide mouthed container

Morning specimen collected 3 consecutive days

Children-gastric washing (tend to swallow sputum)

• Culture

Acid fast staining of sputum-initial test

Culture on Lowenstein-Jensen medium for 8 weeks

Will not grow on Blood Agar plate

BACTEC medium 

Production of radioactive carbon dioxide in 2 weeks

Liquid medium

• Biochemical Reaction

Niacin test

Metabolic by product all mycobacteria

Canary yellow color shows positive

Human tubercle-positive test

Catalase-peroxidase test is positive

• Treatment

Multidrug therapy

Isoniazid (INH), rifampin (6 months)+pyrazinamide (2 months)

3. Mycobacterium leprae

Slender, curved bacilli

Acid fast

Seen singly and in groups

• Important properties

Has not been grow in laboratory/artificial media

Grown in mouse footpad

Human are the natural host

OT is 30 C, grows preferentially in the skin and superficial nerve 

Grows very slowly 

Antibiotic therapy

• Disease

Tuberculoid Leprosy

  • Cell mediated immune (CMI) response to the organism limit it's growth
  • Very few acid fast bacilli seen 

Leprotamous leprosy

  • CMI is poor 
  • Skin and mucous membrane
  • Contain large numbers of organism

• Laboratory Diagnosis

Acid fast stain of skin lesion or nasal scraping

• Treatment

Dapsone 

Combination therapy

Dapsone+rifampin+clofazimine for leprotamous leprosy)

Dapsone+rifampin for Tuberculoid form 

2 years or until the lesion free of organism

4. Spirochetes 

• General characteristics

Elongated, motile (endoflagella-axial filament), spiral bacteria, Gram-negative

Structurally complex

Central protoplasmic cylinder bounded by a cytoplasmic membrane and cell wall of similar structure to that of Gram-negative bacteria

Larger spirochetes are gram-negative, others are too thin to be seen with light microscope

Seen under Dark Ground Microscope

• Classification

Human pathogens belong to following 3 genera 

  • Treponema
  • Leptospira
  • Borrelia

Others (saprophytes) are found in water, sewage and in mouth and genital tracts of humans

5. Treponema pallidum

• Important properties

Thin, delicate with tapering ends 

Have about 10 regular spirals

They are seen only by dark field microscopy, immunofluorescence

Have not been grow on bacteriologic media or cell culture

• Characteristics

Morphology

Thin, 10u with tapering ends

Culture

Do not grow on artificial media

Antigens induce specific antibodies which can be detected by immunofluorescence

• Transmission

From spirochetes-containing lesions of skin or mucous membranes (genitalia, mouth, rectum) of infected person

By intimate contact

Can also transmitted from pregnant woman to their fetus

• Pathogenesis

Produce no toxin or enzyme

Infect endothelium of small blood vessel

Causes Syphilis which can be 

  • Venereal
  • Congenital
  • Non Venereal

Primary lesions


Laboratory Diagnosis

• Microscopy

Dark field microscopy

Direct fluorescent antibody (DFA) test

Not seen on a Gram stain smear

• Non specific serology test

Wasserman complement fixation test

Kahn flocculation test

VDRL (Venereal Disease Research Laboratory)

RPR (Rapid Plasma Reagin) 

• Specific serology test

Using Nichol's strain 

T. Pallidum immobilisation (TPI) test

T. Pallidum hemagglutination test (TPHA)

Fluorescent treponemal Ab absorption test (FTA-ABS)

Very specific, standard reference test 

• Treatment

Penicillin

2nd line

Erythromycin, Tetra/Doxycycline

Neurosyphilis

Ceftriaxone 


010 Actinomycetes & Propionibacterium

1. Actinomycetes

Classification

  • Order: Actinomycetales
  • Shows fungus-like characteristics such as branching in tissues or in culture (look like mycelia). 
    • The filaments frequently segment during growth to produce pleomorphic, diphtheroidal, or club shaped cells. 
  • The cell wall and the internal structures are typical of bacteria rather than fungi.  
  • Some are aerobic and others are anaerobic
  • All are slow growing

The anaerobic genera: Actinomyces, Arachnia, Bifidobacterium

Morphology and cultural characteristics

  • G+ branching, or diphtheroid-like bacilli
  • Anaerobic and require CO2 for growth
  • Non-sporing
  • Will grow on anaerobic BA or PEA.
    • A. israelii, the most commonly isolated species, produces rough, granular colonies that resemble molars.

Biochemistry

  • ID by gas liquid chromatography (GLC) of metabolic by products or fluorescent antibody studies

Clinical significance

  • Are part of the NF found in the cavities of humans and other animals.
  • All may cause actinomycosis or “lumpy jaw” which is a cervicofacial infection that used to occur following tooth extractions or dental surgery which provided traumatized tissue for growth of the microorganism which may also invade the bone.
    • This is rare today because of prophylactic antibiotic therapy.
  • May cause thoracic or abdominal infections
  • May cause meningitis, endocarditis or genital infections

Granules


Actinomyces from Greek "actino" that means mucus and fungus, is a genus of the actinobacteria class of bacteria. They are all Gram-positive and are characterized by contiguous spread, suppurative and granulomatous inflammation, and formation of multiple abscesses and sinus tracts that may discharge sulfur granules. They can be either anaerobic or facultatively anaerobic . Actinomyces species do not form endospores, and, while individual bacteria are rod-shaped, morphologically Actinomyces colonies form fungus-like branched networks of hyphae. 

Biochemistry

  • The organisms are identified based on sugar fermentations and hydrolysis reactions (caseine, tyrosine, etc)

2. Propionibacterium

Classification

  • Two species P. Acnes and P. Granulosome
  • Are described as anaerobic  diphtheroids, though some can grow in CO2.
  • Most clinical isolates are P. acnes which is part of the NF of skin.   

Morphology and cultural characteristics

  • Pleomorphic, small G+B, may have Chinese letter configurations or may be branching.
  • Grow well on CBA, producing tiny translucent to opaque and white to gray colonies.
  • Growth may be slow.
  • Anaerobic, though occasional strains of P. granulosum grow in CO2

Biochemistry

  • Catalase positive
  • Indole +/-
  • Ferment glucose
  • Produce caseinase

Virulence factors

  • Protease

Clinical significance

  • Is part of skin NF
  • Has been implicated in causing acne
    • During adolescence more sebum is produced, and P. acnes metabolizes it to produce fatty acids.
    • These may contribute to the inflammatory response seen in acne.
  • Has also been isolated from joint infections


011 Chlamydia Mycoplasma

1. Chlamydia

Are obligate intracellular parasites

Cell walls are similar to the cell walls of G-B, but lack muramic acid

Have a complex developmental cycle

The infectious form is called an elementary body (EB) which is circular in form and is taken into the cell by induced phagocytosis. 

Inside the phagocytic vesicle replication takes place

• Identification

Direct methods – stain tissues with Giemsa or use a direct fluorescent antibody technique.

The most sensitive method is to culture the organisms in tissue cultures and then stain the infected tissue culture cells

A complement fixation serological test is available as are DNA based tests.

• Virulence factors

Toxicity from attachment and penetration

• Clinical significance

Chlamydia trachomatis – serotypes A-K and L1,2,3; the serotype determines the clinical manifestation.

Genital tract infection (serotypes D-K) – is the major cause of non gonococcal urethritis; is sexually transmitted and frequently found concomitantly with N. gonorrhoeae

In males symptoms include urethritis, dysuria and it sometimes progresses to epididymitis

In females symptoms include mucopurulent cervical inflammation which can progress to salpingitis and PID.

Inclusion conjunctivitis – this occurs in both newborns and adults and a genital tract infection is the source of the infection (serotypes D-K); is a benign, self-limited conjunctivitis which heals with no scarring

• Clinical Syndromes

1. Trachoma

Chronic infection or repeated infection

inflammation and follicle formation involving the entire conjunctiva

Lymphogranuloma venereum (serotypes L1, 2, 3) is a venereal disease that occurs in poor, tropical areas.

Upon infection, widespread dissemination takes place and a primary, painless lesion (either a vesicle or an ulcer) occurs at the site of entry within a few days.

This heals with no scarring.

A secondary stage occurs 2-6 weeks later with symptoms of regional suppurative lymphadenopathy (buboes) that may drain for a long time and be accompanied by fever and chills.

Arthritis, conjunctival, and CNS symptoms may also occur.

A tertiary stage may occur and is called the urethrogenital perineal syndrome.

This is characterized by structural changes such as non-destructive elephantiasis of the genitals and rectal stenosis.

• Laboratory diagnosis

1. Cytology

Examination of stained cell scrapings for the presence of inclusion bodies

2. Culture

the most specific method for diagnosis

cultures of susceptible cells

iodine-staining inclusion bodies

3. Serology

Detection of high titer IgM antibodies is indicative of a recent infection

2. Chlamydia a life cycle


3. Mycoplasma

2 medically important genera:

Mycoplasma

Ureoplasma

Three common clinical isolates – M. pneumoniae, M. hominis, and U. urealyticum

• Morphology and cultural characteristics

Do not possess the distinctive cell wall of bacteria

Plasma membrane is the outermost part of the organism and is unique in bacteria in that it has a high content of sterols that act to prevent osmotic lysis

Very small in size (too small to be seen with an ordinary light microscope) and highly pleomorphic

Don’t stain with a Gram’s stain

Non-motile

May possess a capsule

Although some are free living, most are closely adapted parasites

Grow on media enriched with serum (need cholesterol)

Grow beat at 35-370 C either aerobically or anaerobically

M. pneumoniae grows in 5-14 days, M. hominis in 2-4 days, and U. urealyticum in 24-28 hours.

M. pneumoniae colonies resemble fried eggs and can be stained with Dienes stain (they stain blue)

• Identification

M. pneumoniae

Isolation in culture

Ability of colonies to hemolyze guinea pig RBCs

Rise in specific antibody titer

Cold agglutinin test – a nonspecific test in which the patient produces cold reacting antibodies that agglutinate type O human RBCs at 40 C, but not at 370 C

A single titer of 1:128 is significant and occurs in 7 days and disappears in 6 weeks.

• M. hominis

Isolation in culture

No hemolysis of guinea pig RBCs

• U. urealyticum

Urease production

• Virulence factors

Not invasive and simply colonize cell surfaces through specific binding

Damage to host tissues may be due to toxic metabolic products

• Clinical significance

M. pneumonia – the major cause of primary, atypical pneumonia (walking pneumonia)

Transmitted by droplet infection

Genital tract infections - caused by M. hominis and U. ureolyticum which may also be found as part of the NF in the genital tract

May cause nongonococcal urethritis, PID, post-partum fever, infertility, stillbirth, spontaneous abortion, and acute urethral syndrome


Quiz 1 notes

Bacterial Taxonomy

Gram staining

Biochemical Testing

Microscopy

Catalase

Coagulase

AST (novobiocin) Staphylococcus saprophyticus resistant, Staphylococcus epidermidis sensitive

Lancefield Group & Hemolysis Streptococcus

Biochemical Reaction


Quiz 2 notes

Shigella boydii is a glucose fermenting org

Skirrow's campylobacter

Gastric ulcers helicobacter short curved spiral gram -ve Choco Agar 

Vibrio culture TCBS not Loeffler's serum transport medium Venkataraman Ramakrishnan 

Swarming Proteus glucose fermentor

Enterobacteriacaea not oxidase positive

Shigella boydii cause bacillary dysentery

Salmonella Mac

Klebsiella gram -ve short non motile capsulated TSI glucose lactose

Salmonella enterica produce H2S

Oxidase positive Pseudomonas

Non motile clear non lactose fermenting not produce H2S Shigella


Quiz 3 notes

Mycobacterium tuberculosis, acid-fast staining prepare smear of sputum suspected, Direct smear: ZN, droplet, × Blood Agar, sputum sample

Mycobacterium leprae, Leprotamous leprosy, Tuberculoid leprosy

Mycobacterium, × facultative anaerobe, ✓ obligate aerobe

VDRL, syphilis, Treponema pallidum, RPR, Dark field microscopy, transmission: intimate contact

Neisseria gonorrhoeae grow on NYC, glucose

Dark ground microscopy, Leptospira interrogans, spiral, × Gram-positive

Haemophilus, haemin and nicotinamide adenine dinucleotide (Choco Agar), 

Haemophilus ducreyi, requires X factor

Bacillus anthracis, bamboo appearance, non hemolytic on 5% Blood Agar, gram-positive square end, non motile, antiphagocytic capsule D-glutamate

Actinomycetes, hyphae, endospores anaerobic, diphteroid-like bacilli

Mycoplasma pneumoniae, droplet

Cornyebacterium diphtheriae, Chinese pattern, Albert's stain for metachromatic granules, Tellurite Blood Agar, transmitted by airborne droplet





SUMMARY 

Chapter 1

Taxonomy is the practice and science of classification. 

Family: (a group of related) genera

Genus: species

Species: strains

Type: a set of strain within a species

Strain: single isolate of a particular species

Decolorization fail, retain purple. 

1. Isolation and observe growth in pure culture

  • streaking
  • colonies visible to naked eye appear after incubation 
  • size, texture, color, hemolysis  

2. Microscopic examination

  • gram stained

3. Species identification

  • bacteria are speciated using isolated colonies-add 24 hrs
  • generally use physiological tests

4. AST

  • susceptible to specific antibodies

Gram positive purple/blue, Gram negative pink

Lab temperature 37

OF glucose, oxidative & fermentative, bromthymol blue green turns yellow fermentative 

Simmon citrate, utilize citrate as carbon source, bromthymol blue indicator green turns Prussian blue

Phenol red fermentation broth, acid and gas production from variety of sugars, phenol red indicator turns yellow (sugar fermented, acid) 

Urease, identify Proteus from non-lactose fermenting enterics, phenol red turns deep pink (alkaline ammonia)

H2S, SIM agar is used, insoluble black precipitate is positive (ferrous ammonium sulfate) 

Phenylaline deaminase, deaminate phenylaline into phenylpyruvic acid, dark green (ferric chloride react with phenylpyruvic acid) 

TSI, H2S production as well as carbohydrate fermentation

Indole, SIM agar with tryptophan, cherry red reagent ring is positive 

MRVP, produce acid or neuralize into acetoin, MR red positive, VP pink positive

Chapter 2 

Gram+, non sporing, non motile, occurs in singly, pairs and in irregular clusters, opaque colonies, facultative anaerobes, salt tolerant, highly resistant to drying, hemolytic

Staphylococcus aureus, coagulase/DNAse +, opportunistic, NF of skin, food poisoning, scalded skin, toxic shock syndrome

Staphylococcus epidermidis, sensitive to novobiocin, NF of skin, endocarditis, catheter

Staphylococcus saprophyticus, resistant to novobiocin, cyctitis, pyelonephritis, urethritis

Micrococci, gram +

Specimen collection: wound, lesion, pus, sputum

Culture: Blood Agar, MSA (selective & differential)

Chapter 3

Gram+, pairs/chains

Streptococcus pyogenes, group A, beta hemolysis, bacitracin +, PYR + (red)

Streptococcus agalactiae, group B, beta hemolysis, bacitracin -, CAMP + for group B

Streptococcus enterococci, group D, gamma hemolysis, 40% bile esculin + (black halo, fluorescence UV), PYR +

Streptococcus pneumoniae, alpha hemolysis, bile solubility & optochin +

Streptococcus viridans, alpha hemolysis

Lancefeld, proteins on cell wall

Alpha, partial lysis of RBC, greenish discoloration 

Beta, complete lysis, clearing of blood, grey

Gamma, no hemolysis

Catalase, staphy +, strep -

Chapter 4 

opportunistic=cause disease under certain condition/in certain hosts

primary=in anyone

facultative anaerobes, ferment glucose, non spore forming, none have cytochrome oxidase, contain endotoxin in cell wall

E. coli, primary & opportunistic, NF of GI, UTI, septicemia, neonatal meningitis, MacConkey red (lactose+), no H2S 

Enterobacter, opportunistic MacConkey red, no H2S, motile

Proteus, opportuinistic swarming growth, gram -, non lactose, nosocomial UTI, oxidase -

Klebsiella, primary & opportunistic, gram -, non motile, capsulate, MacConkey red, no H2S, Fried-Lander pneumonia, neonatal meningitis, lung abscess, wound infection, UTI

Serratia, opportunistic hospital acquired infections, nosocomial & bacteremic outbreaks, TSA pink to red, prodigiosin, ferments lactose slowly, resistant to multiple antibiotics 

Chapter 5 Primary pathogen  

Salmonella spp, gram -, facultative anaerobes, non lactose, most produce H2S, enterocolitis, septicemia, enteric fever

Salmonella enteritidis ser typhi, produce H2S and gas

Salmonalle typhi, no gas

Salmonella bongori

Shigella, dysentery, non motile, no H2S, fragile, non lactose, O antigen

Shigella dysenteriae, group A

Shigella flexneri, group B

Shigella boydii, group C, no H2S and gas

Shigella sonnei, group D

Mac, CLED, XLD

Chapter 6 

Campylobacter jejuni/coli, gram -, motile, slender spiral, microaerophilic (5%), grow at 42~43, oxidase+, Blood Agar, mucoid colonies, pleomorphic, Skirrow's, stool, diarrhea

Helicobacter pylori, gram -, spiral, biochemically inactive, motile unipolar, non capsulated, produces urease, catalase and oxidase, microaerophilic, needs CO2, Warthin-starry silver stain, H&E, choco & Skirrow's, Biopsy Urease -placed in urea contain pH indicator -positve within 2m~2h, Urea Breath (urea C13/14) positive if isotope gives high reading in CO2

Vibrio, gram -, unipolar, curved/rod, non sporing & capsulated, Mac (colorless then pink), Blood (no hemolysis), Nutrient (moist, round discs), ferment glucose, mannitol, sucrose, maltose with acid production, non lactose, catalase & oxidase +

TCBS: cholera yellow (sucrose), parahaemolyticus (8% NaCl) & vulnificus green

Transport medium: Venkatraman-Ramakrishnan, Cary-Blair (shigella & salmonella), Alkaline peptone water 

Chapter 7 

Neisseria, gram -, oxidase +, sensitive to fatty acids & trace metals, growth on Choco Agar 37

N. gonnorrhoeae, diplococci, fragile, gonococcus, NYC +, glucose

N. meningitidis, NYC+, glucose, maltose

Moraxella catarrhalis, oxidase +, NYC -, x sugar fermentation

Actinetobacter, oxidase-, catalase+

Haemophilus, small, gram-, rod or coccobacilli, catalase&oxidase+, glucose+, convex, smooth, pale, grey, transparent colonies

growth factor VX

X haemin , ducreyi

V nicotiamide adenine dinucleotide, parainfluenzae

XV, influenzae, aegyptius

Specime collection: CSF, blood, throat swab, sputum, pus, never be refrigerated

Brucella, non capsulated, B. melitensis (goats and sheeps), B. abortus (cattle), B. suis (pigs), Brucellosis, slaughterhouse case, grow slow on Blood Agar, Choco Agar, produce urease, H2S, fail to grow on EMB, MacConkey, need CO2

Moraxella, aerobic, oxidase +, diplococcus, rarely caused infections in humans 

Chapter 8

Bacillus anthracis, anthrax, gram + rod with square end, chains, bamboo appearance, antiphagocytic capsule is composed of D glutamate, cutaneous, GI, pulmonary, non hemolytic on Blood Agar (greyish white)

Cornyebacterium, free living saprophyte, gram + rod, Chinese letter, airborne droplet, tropic, Blood Agar (rapid growth 6~8 hrs), Tellurite Blood Agar (grey/black colonies), Loeffler's serum slope, Albert's stain for metachromatic granules.  

Listeria monocytogens, gram+. cold temp, meningitis, sepsis, resembling diphteroids, motile, small grey colonies (beta-hemolysis)

Chapter 9

Mycobacterium TB, Koch's Bacillus, acid-fast, fungus-like, obligate aerobe, resistant to acids, alkalis, dehydration, discoloration, non motile, non capsulated, non sporing, slow growing, airborne droplet, sputum, Lowenstein-Jensen medium (8 w), no growth on Blood Agar, BACTEC (2 w, produce radioactive CO2), liquid medium, catalase/oxidase +, Niacin (canary yellow)

Mycobaterium laprea, not grown in artificial media, grow in mouse footpad, slow growing, Tuberculoid leprosy, Leprotamous leprosy

Spirochetes, elongated, motile (endoflagella), spiral, gram -, dark ground microscopy

Treponema pallidum, immunofluorescence, dark field, tapering ends, intimate contact, syphilis (nevereal, nonvenereal, congenital), Direct Fluorescent Antibody, Wasserman complement fixation test, Kahn flocculation, VDRL, RPR, Nichol's strain, TPImmobilasation, TPHaemaAgglutination, FluoresecentTAntibody-ABSorption, penicillin, ceftriaxone, erythromycin

Chapter 10

Actinomycetes, gram+, fungus-like (mycelia) (hyphae), pleomorphic, diphteroid, sluc shaped, some aerobic others anaerobic, slow growing, non sporing, NF in cavities, actinomycosis (lumpy jaw), thoracic, abdominal, meningitis, endocarditis, genital infections, granulomatous inflammation, no endospores

Actinomyces, Arachnia, Bifidobacterium, A. israelii

Propionibacterium, gram+, anaerobic diphteroids, pleomorphic, Chinese letter, grow well on CBA (translucent to opaque/white to grey colonies), slow growing, catalase+, indole+/-, ferment glucose, produce caseinase, protease as virulence factor, isolated from joint infections

P. acnes NF of skin, P. granulosum

Chapter 11

Chlamydia, obligate intracellular parasite, cell wall lack muramic acid, Giemsa stain, DFA, trachomatis, genital tract infection, inclusion conjunctivitis (eye), lymphogranuloma venereum

Mycoplasma, small pleomorphic, non motile, may posses a capsule, free living/adapted parasites, Diene's stain (fried egg), hemolyze guinea pig RBC, Cold agglutinin test

M. pneumoniae (droplet), M. hominis (no hemolysis, NF GI), Ureoplasma urelyticum (produce urease, NF GI)

Comments

Popular posts from this blog

My MLT Notes 2

MLT     以下是我个人从网上搜来的一些与医学知识有关的网站以及平台。这些都是link。 1. Human Anatomy & Physiology https://med.libretexts.org/Bookshelves https://opentextbc.ca/anatomyandphysiology/chapter/introduction-4/ https://courses.lumenlearning.com/boundless-ap/front-matter/download-lecture-slides/ 2. Biochemistry http://edusanjalbiochemist.blogspot.com/?m=1 https://science-pdf.com/category/books/medicine/biochemistry/?fbclid=IwAR3LQPHvcKO2cvI7X_kYu3UsjqbjlyFnaE1TsBk0H-e5Nj533wdyy2gmQes 3. Immunology https://www.slideshare.net/mobile/hmirzaeee/basic-immunology http://www.biology.arizona.edu/immunology/tutorials/immunology/main.html 4. MLT http://image.bloodline.net/category.html https://www.google.com/amp/s/www.proprofs.com/quiz-school/topic/amp/mlt https://www.studystack.com/LaboratoryScience https://www.scribd.com/document/305391444/AIMS-Professional-Examination-Pack 5. Medic https://geekymedics.com/ http://www.freebookcentre.net/medical_text_books_journals/medical_text_books_online.html https://en.m.w...

My License

  考驾照还真是难呢!对我这个连车子的方向盘都没有碰过的人来说,这是一个艰难的过程。更何况,我还要考传说中的Manual车。   那么先从头来复习考驾照的过程吧! 1. KPP 01:6hrs kursus + ujian bhg 1 很简单的笔试,当时拿了47/50就过关了。L牌并不难考,只要你肯读书。交了钱,几天后就能拿到L牌驾照咯! 2. KPP 02:5.5hrs amali di litar 最好学会驾车才去。这项需要你会的努力和坚持不懈的学习驾车。虽然都是基本的练习,但却需要熟练。 3. KPP 03:10hrs amali di jln raya 这里就要考验你的驾车技术以及安全意识了。路上行驶不好还会被教官骂的呢! 4. Penilaian pra ujian 30min 这是JPJ考试前都需要做的。确保你有能力参与JPJ考试。过关了自然就能考P牌,没过就要从KPP02重新开始。 5. JPJ test bhg 2&3 只要过关就能拿P牌了。两年后,P牌驾照可更换成C牌。   为了学车,付出劳力汗水财物,我简直是......累死了。再来一次......不!千万别再来一次了。我只想好好过日子,乖乖地宅在家里了。   情势所迫,我还是得握有驾照在手,不管怎么样,no pain, no gain

My Lab Notes

My Lab Notes 1 Laboratory procedure and instrumentation Answer these questions. Introduction to medical laboratory procedure and instrumentation  1. Define medical laboratory. 2. Determine the importance of medical laboratory. 3. Determine the units in medical laboratory. 4. Define medical laboratory technologist. 5. Determine the forms of hazards. 6. Determine personal protective equipment (PPE). 7. 8 safety equipments. 8. Explain practice good personal hygiene. 9. Draw and name hazards. 10. Explain about the ethics. Glassware and plastic ware 1. Type of glassware. 2. Name, function, and the form of glassware. 3. Explain about cleaning laboratory glassware. Medical terminology 1. Pericarditis. 2. Structure of medical terms. 3. Abbreviation and acronym. 4. Miscellaneous. Metric system 1. Explain about the importance of measurement. 2. List out the international systems of units (SI). 3. Conversion factors. Basic laboratory instruments General lab equipment 1.0 1. Name, principle, t...