Clinical Microbiology Bacteriology

MEDICAL MICROBIOLOGY: BACTERIOLOGY
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STAPHYLOCOCCI
Staphylococcus aureus
Clinical importance:
Upper respiratory tract infections, gastrointestinal tract infections, genital tract infections, impetigo,
cellulitis, bacteremia, osteomyelitis, toxic shock syndrome, wounds, burns, food poisoning,
endocarditis.
Specimens:
Sputum, urine, nose, ear, eye, bedsores, vaginal swabs, blood cultures, body fluids, CSF, stool, food.
Microscopic examination:
Gram positive cocci in clusters.
Culture:
Facultative anaerobic.
370C (Body temp.) optimal temperature.
Blood agar.
Nutrient agar.
Mannitol agar.
Tpey agar.
MacConkey agar without crystal violet.
Coagulation test: positive.
DNAse: positive .
Catalase test: positive.
Novobiocin: sensitive.
Haemolysis: haemolytic.
Susceptability testing: Penicillin.

Erythromycin.
Cloxicillin.
Bactrim.
Augmentin.
Clindamycin.
Flucidin.
Amikacin.
Vancomycin.
Staphylococcus epidermidis
Clinical aspects:
Urinary tract infections (hospital acquired), osteomyelitis (post operative), endocarditis (prosthetic
valve), indwelling devices (catheters, prosthetic joints), bacteremia (neonates), endophthalmitis.
Specimens:
Urines, blood cultures, CSF, body fluids.
Culture:
370C (body temp.) optimum temperature.
Blood agar.
Nutrient agar.
Mannitol salt agar.
Coagulase: negative.
DNAse: negative.
Catalase: positive.
Novobiocin: sensitive.
Haemolysis: non-haemolytic.
Susceptibility testing: see notes for Staph. aureus.
Staphylococcus saprofiticus
Clinical aspects:
UTI in young females.
Specimens:
Urine.
Culture:
370C optimum temperature.
Blood agar.
Nutrient agar.
Cled agar.
Coagulase: negative.
Dnase: negative.
Catalase: positive.
Novobiocin: resistant.
Haemolysis: non-haemolytic.
Susceptibility testing: See notes for Staph. aureus.

STREPTOCOCCI
● Lancefield group A, B and D.

● Viridans spp.
● Pneumococci.
Streps can be differentiated by: beta-haemolysis and alpha-haemolysis.

Beta-haemolytic: These strains produce a soluble haemolysin.
Causes a clear zone of haemolysis around colonies on blood agar. (100 %)
Alpha-haemolytic: Some species do not produce a soluble haemolysin.
Causes partial haemolysis around the colonies creating a green color on blood
agar.
Non-haemolytic: Non-haemolytic strains do not produce soluble haemolysin and will not have a
zone of haemolysis around the colonies.
Streps microscopically: Gram positive cocci in chains.
Streptococcus pyogenes (Group A) (β−hemolytic)
Clinical aspects:
Sore throat, scarlet fever, cellulitis, impetigo, vaginitis, otitis media, arthritis, wound infections;
indirectly glomerulo-nephritis and rheumatic fever.
Specimens:
Throat swabs, wound swabs, vaginal swabs, blood cultures, sputum, urine.
Culture:
Blood agar with bacitracin disk: sensitive to bacitracin.
Bile-aesculin agar: no growth.
6.5% NaCl: negative.
Catalase test: negative.
Susceptibility testing: Penicillin.

Erythromycin.
Streptococcus agalactiae (Group B) (β−hemolytic)
Clinical aspects:
Neonatal meningitis, neonatal septicaemia, endocarditis, cellulitis, arthritis, genetial tract, bovine
mastitis.
Specimens:
Urine, blood culture, sputum, vaginal swabs, wound swabs.
Culture:
Blood agar with bacitracin disc – not sensitive.
Bile aesculin agar: growth but no hydrolysis of aesculin.
6.5 % NaCl: negative.
Catalase test: negative.
Camp test: positive.
Erythromycin.
Streptococcus (Enterococcus) faecalis (Group D) (variable hemolysis)
Clinical aspects:
UTI, subacute bacterial endocarditis, ear infections, biliary tract infections, supperative abdominal
lesions. Most enterococcal infections by E.faecalis. E.faecium resposible for most of the rest –
around 5 – 10 %.
Specimens:
Urine, wound swabs, ear swabs, blood cultures.
Gram positive cocci ovoid pairs or short chains,
Culture:
Blood agar with bacitracin – resistant.
Bile aesculin agar: hydrolysis (black).
6.5 % NaCl: positive (yellow).
Catalase: negative.
Enterococcus agar: small pink colonies.
Susceptibility testing: Penicillin 8.

Vancomycin.
Nitrofurantion (urine).
High level of antibiotic resistance – plasmid mediated; can be transferred to other strains.
Strepex kit test can be used for grouping of streps. (enzyme extraction.)
Serology: ASOT (Antistreptolysin O titre.)
NB: http://en.wikipedia.org/wiki/Enterococcus
Streptococcus viridans (α -strep)
Species:
S. mutans, S. mitis, S. milleri, S. sanguis, S. bovis I, S. bovis II.
Clinical aspects:
Dental sepsis, dental caries, subacute bacterial endocarditis.
Specimens:
Throat swabs, sputum, blood culture.
Gram positive cocci in chains or ovoid.
Culture:
37oC (Body temperature) optimum temperature.
Blood agar with penicillin, bacitracin.
Bile aesculin agar: different for specific species.
6.5 % NaCl: different for specific species.
Biochemical reactions: different for specific species.
Erythromycin.
Streptococcus pneumoniae (Pneumococcus) (α−strep)
Clinical aspects:
Lobar pneumonia, acute and chronic bronchitis, bronchopneumonia, otitis media, sinusitis,
meningitis, peritonitis, arthritis, conjunctivitis.
Specimens:
Sputum, pus swabs, blood cultures, csf.
Gram positive lanceolated diplococcus.
Culture:
370C (body temperature) optimum.
5 – 10 % CO2.
Blood agar with optochin: sensitive to optochin, draughtman colonies with coloration.
Mueller Hinton agar with oxicillin disc.
Bile solubility: bile soluble.
Antigenic characteristics:
Serotyping can be done, or a capsule swelling test Quelling reaction.

Erythromycin.
Tetracyclines.
Ceftriaxome.
Chloramphenicol.
Strep Q and A.
NEISSERIA
Neisseria gonorrhoea (Gonococci)
Clinical aspects:
Venereal disease, transmitted by direct, close sexual contact, acute gonorrhoea – males: urethra, and
cervix uteri, blockage and scarring of fallopian tubes (salpingitis), anorectal and oropharyngeal –
homosexuals, conjunctivitis – newborn infants, arthritis, meningitis.
Specimens:
Urethral swabs, eye swabs, blood culture, csf, sputum, urine, joint fluid, nasopharyngeal swabs.
Specimen transport:
Stuart's transport media; survive 6 – 12 hours.
Gram negative diplococci. Located intracellularly in polymorphonuclear leucocytes.
Culture:
Blood agar CO2.
Chocolate agar CO2.
Thayer Martin agar CO2.
New York City agar CO2.
350 - 370 C (+/_ body temperature), 5 – 10 % CO2.
Oxidase test: positive.
Beta-lactamase test: positive or negative.
Cystine-triptic digest agar (CTA sugars) – control, glucose, maltose, lactose, sucrose; will only take
glucose.

Ciprofloxacin.
Ceftriaxone.
Tetracycline.
Ofloxacin.
Neisseria meningitidis (Meningococcus)
Clinical aspects:
Acute purulent meningitis, septicaemia with petechial rash.
Specimens:
Csf, blood culture, skin lesions, nasopharyngial swabs.
Gram negative diplococci. Intra-cellular or extra-cellular.
Culture:
Blood agar CO2.
Chocolate agar.
Thayer Martin agar CO2.
New York City agar CO2.
35 – 370C, 5 – 10 % CO2.
Beta lactamase test: positive or negative.
CTA sugars: glucose and maltose positive.

Ceftriaxone.
Chloramphenicol.
Neisseria lactamica
Clinical aspects:
Primary habitat the nasopharynx of children (3m – 6 years), can cause meningitis, bacteremia.
Rarely from genital tract.
Specimens:
Csf, blood cultures, vaginal swabs.
Gram negative cocci.
Culture:
Same media as for N. meningitidis.
Will grow on nutrient agar at 22oC (Room temperature).
Glucose: positive, maltose: positive, lactose: positive.
ONPG: positive.
Serological cross agglutination between N. meningitidis and N. lactamica.
Moraxella catarrhalis
Gram negative, oxidase positive diplococcus.
Clinical aspects:
Saprophytic organism of the upper respiratory tract, meningitis, bacteremia in immunosuppressed
patients.
Specimens:
Sputum, throat swabs, body fluids, tracheal aspirates.
Culture:
Grow at RT (22oC).
Ordinary media without serum.
CTA sugars: no fermentative properties.
Susceptibility testing: Cotrimoxazole.

Co-amoxyclav.
Amoxicillin.
Ciprofloxacin.
Ceftriaxone.
Tetracycline.
CORYNEBACTERIUM
Corynebacterium diphtheriae
Clinical aspects:
Diphtheria – a human pathogen, produces potent exotoxin with an affinity for heart muscle and
peripheral nerves.
Inflammatory lesion with membranous exudate affecting the mucosa of the upper respiratory tract.
Wound infections, skin infections.
Specimens:
Throat, nose and wound swabs.
Pleomorphic gram positive rods, chinese lettering (coryneforms). Special stains to demonstrate the
volutin granules – Neisser and Alberts stain.
(Remember: Cocci are generally gram positive, except Neisseria and Moraxella.
Bacilli are generally gram negative (GNB), except Corynebacteria, Listeria, Clostridia
(anaerobes), acid fast and some others.)
Culture:
Optimum temperature 370C (Body temp).
Grows best on medium containing blood or serum, eg. Loeffler serum, Tellurite agar (Hoyles
medium), Chocolate agar.
CTA sugars: ferment glucose and maltose.
Nitrate: positive.
Urease: positive.
Catalase: positive. (NB)
Elek plate: agar gel precipititation test for toxigenicity of C. diphtheriae.

Erythromycin.
Do these sensitivities on Muller Hinton agar.
Corynebacterium ulcerans
Clinical aspects:
Animal parasite, cause mastitis in cattle, spread to man by raw milk consumption, ulcerative
exudative throat infections, cardiac and neurological complications.
Gram positive pleomorphic bacilli.
Culture:
Blood agar – small zone of haemolysis.
Loeffler serum.
CTA sugars: ferments glucose and maltose.
Nitrate: negative.
Urease: positive.
Catalase: positive.

Erythromycin.
Lysteria monocytogenes
Clinical aspects:
Meningo-encephalitis in people, listeriosis, food outbreaks eg. chicken.
Pregnancy infections eg. septic abortion, neonatal sepsis (granulomatosis infantiseptica), cerebritis
(brain abscesses, tumor, stroke), sepsis.
Specimens:
CSF, blood culture, liver tissue, faeces, food, exudates.
Gram positive bacilli straight or slightly curved.
Gram negative in older cultures.
Non-spore forming.
Culture:
Aerobic and facultative anaerobic.
35 – 37 0C (body temperature) optimum temperature,
Blood agar (sheep blood): small translucent with a narrow zone of complete haemolysis.
Ordinary media, but better on media containing serum blood or liver extract.
Catalase: positive.
Motility at 250C (RT) – tumbling or head-over-heals.
Esculin: hydrolysis.
CTA sugars: ferment glucose, maltose and salicin promptly.
Camp test: positive but a rectangular rather than an arrow head shaped zone of haemolysis.

Ampicillin.
MYCOBACTERIUM
Mycobacterium tuberculosis
Clinical aspects:
Cause tuberculosis, chronic granulomatous lesions.
Specimens:
Sputum, laryngeal swabs, gastric lavage, bronchoscopic specimens, urine, pleural and peritoneal
fluids, csf, pus, tissue.
Gram positive bacilli, but because of the waxy material in the cell wall M. tuberculosis requires a
mordant dye in the presence of heat; Ziehl-Neelsen stain, or use a fluorocrome stain – Auramine
stain.
TB is acid and alcohol fast, bacilli stain red against a blue background.
We record our results according to the IUAT (International Units Against TB):
Negative No acid and alcohol fast bacilli.
Scanty positive 1 – 10 bacilli per smear.
1+ positive 10 – 100 bacilli per smear.
2+ positive 1 – 10 bacilli per field.
3+ positive more than 10 bacilli per field.
Culture:
Strict aerobe.
Optimum temperature: 35 – 37 0C (Body temperature).
Loewenstein-Jensen media: raised, dry, wrinkled. At first whitish, later buff colored or yellow.
Blood agar.
Serum agar.
Media which contain whole egg or vegg yolk.
Catalase: positive.
Niacin test: positive.
Nitrate reduction: positive.
Susceptibility testing: INH.

Rifampicin.
Ethambutal.
See also:
http://en.wikipedia.org/wiki/MDR_TB
http://en.wikipedia.org/wiki/XDR_TB
Mycobacterium leprae
Clinical aspects:
Cause leprosy – affects skin, mucous membranes.
Specimens:
Sputum, scrapings from nasal mucosa, ulcerated nodule on skin. Scraping of tissue after skin
incision – particularly of ear lobe.
Stain with modified ZN using 20 % H2SO4 as decolorant.
Long slender bright red bacilli arranged like match bundles (globi or globus shape).
HAEMOPHILUS
Haemophilus influenza
Clinical aspects:
Meningitis, laryngoepiglottitis (croup), otitis media, sinusitis, upper respiratory tract infections,
pneumonia, bacteremia, endocarditis, urogenital, maternal and perinatal infections.
Specimens:
Csf, sputum, ear swabs, throat swabs, eye swabs, blood cultures, vaginal swabs, urethral swabs,
amniotic fluid, placental tissue.
Small gram negative pleomorphic bacilli.
Culture:
Aerobic.
Requires both X and V factors.
X-factor is heat resistant, present in haemin.
V-factor is a respiratory coenzyme destroyed by heat at 1200C, but released from blood cells by
heating at 700 – 800C (chocolate agar).
Blood agar with Staph. streak: blood agar provide X-factor; Staph. hemolyses the RBC to release
the V-factor.
Colonies form sattelism around the Staph. streak.
Nutrient agar with Staph. streak: no growth.
Catalase: positive.
Oxidase: positive – slow reaction.
Beta-lactamase: positive or negative.

Co-amoxyclave (Augmentin).
Amoxicillin.
Ciprofloxacin.
Ceftriaxone.
Tetracycline.
Do these sensitivities on Haemophilus Test Medium (HTM) – has X and V without blood.
Haemophilus parainfluenza
Clinical aspects:
Endocarditis, meningitis, brain abscesses, peritonitis, purulent urethritis.
Otherwise a normal commensal in the throat and mouth.
Specimens: As for Hflu.
Microscopic examination: As Hflu.
Culture:
Use same media as for Hflu.
Requires only V-factor for growth – grow on blood agar with Staph. streak and also on nutrient agar
with Staph streak (Does not require X factor.).
Colonies are bigger and more opaque than Hflu.
On MH does not grow around X, as it only requires V factor for growth) – only method to
differentiate from Hflu (as at 2003).
Susceptibility testing: As for Hflu.
ENTEROBACTERIACEAE
Properties of enterobacteriaceae: gram negative bacilli; grow in presence of bile salts (MacConkey
agar), ferment glucose aerobically or anaerobically; reduce nitrates to nitrites, oxidase negative;
non-sporing, motile or non-motile; catalase positive.
Includes: E. coli, Klebsiella, Enterobacter spp., Serratia, Proteus, Providencia, Citrobacter,
Salmonella, Shigella, Yersinia.
Natural habitats: widely distributed on plants and in soil, water and intestines of humans and
animals.
S. typhi species only found in man – typhoid fever.
Klebs. pneumonia can be found in the environment but can also cause urinary tract infection (UTI)
and upper respiratory tract infection (URTI).
● COLIFORM BACILLI
1. Escherichia coli
Clinical aspects:
UTI-cystitis, pyelonephritis, gastro-enteritis, respiratory tract infections, neonatal meningitis,
endotoxic shock.
Specimens:
Urine, stool, sputum, csf, wound and pus swabs, water.
Gram negative bacilli, non-sporing.
Culture:
Aerobic and anaerobic.
370C. (body temperature) optimum.
MacConkey agar: lactose fermenter – smooth, rose-pink in color.
CLED agar: bright pink in color.
Blood agar: can be haemolytic.
Biochemical reactions:
Lactose: fermenter.
Motile.
Indole: positive.
Mannitol: fermenter.
Forms gas from glucose.
Citrate: negative.
KCN: negative.
Methyl red: positive.
Voges Proskauer: negative.
Public health:
Do IMVEC for drinking water supplies: Indole positive, MR positive, VP negative, citrate negative.
Eijkman test: ability to form gas at 440C from lactose.

Co-amoxyclav. (Augmentin)
Amikacin.
Amoxicillin.
Gentamycin.
Ciproflox / Ofloxacin.
Ceftriaxone.
Nitrofurantoin. (urine)
Naladixic acid. (urine)
Cefoxitin.
Antigenic structure: Serotyping done.

E. coli in diarrhoea:
Enteropathogenic strains (EPEC)
Enterotoxogenic strains (ETEC)
Enteroinvasive strains (EIEC)
Enterohaemorrhagic strains (EHEC).
2. Klebsiella species
Clinical aspects:
UTI, URTI – pneumonia, bacteremia.
Specimens:
Urine, stool, sputum, blood cultures, wound, pus and burn swabs.
Gram negative bacilli, non-sporing.
Culture:
MacConkey agar: pink mucoid colonies.
CLED agar: light pink mucoid colonies.
Blood agar: large grey mucoid colonies.
Lactose fermenter.
Non-motile.
Indole negative. Klebs. oxytoca is indole positive.
Mannitol: fermenter.
Forms gas from glucose.
Citrate: positive. (remember E.coli is neg.)
KCN positive. (E.coli neg.)
Methyl red: negative. (E.coli and others pos.)
Capsulated: can do capsule swelling test with specific antisera for klebsiella.
Susceptibility testing: as for E.coli.
3. Enterobacter spp.
Clinical aspects:
Opportunistic pathogens, they are frequent colonizers of hospitalized patients – specifically those
on antibiotics, burn wound, urinary and respiratory infections.
Gram negative bacilli.
Culture:
Very similar to Klebsiella spp.
Motile.
MacConkey agar: non-lactose fermenting. (late lactose fermenting.)
ONPG: positive. (late lactose fermenters.)
Susceptibility testing: as for E.coli.

Enterobacters are usually resistant to first generation cephalosporins.
● PROTEUS MIRABILIS
Clinical aspects:
UTI, septicemia, wound infections.
Specimens:
Urine, blood culture, csf, wound and pus swabs.
Gram negative bacilli, coccal forms to long filaments.
Culture:
Blood agar: fishy smell and swarming over plate.
MacConkey agar: non-lactose fermenting colonies.
CLED agar: non-lactose fermenting (transparent colonies).
Stop swarming-PNPG: 0.215 g p-nitrophenylglycerol to 500 ml media, 4% agar, MacConkey agar
or DCA.
Non lactose fermenter. (NLF)
Urease positive. (Singers turn blue.)
Motile.
Indole negative.(Note: other Proteus spp. and Providencia and Morganella are indole positive.)
Mannitol negative.
H2S positive.
Gas from glucose.
Citrate positive.
KCN positive.
PPA positive. (Phenyl-alanine agar.)
Methyl red: positive.
Voges proskauer: positive or negative.
Antigenic structure:
P.mirabilis can be serotyped by their O-antigens into 49 O-groups
Susceptibility testing: same as for E.coli.

Resistant to Nitrofurantion in urines.(differentiate E.coli.)
● SALMONELLA spp.
Clinical aspects:
Diarrhoea, food poisoning, septicemia.
Specimens:
Stool, vomit, foodstuffs, blood cultures.
Culture:
Blood agar: grey, smooth colonies.
Nutrient agar: colorless smooth colonies.
MacConkey agar: non lactose fermenting.
SS agar: nlf with H2S (black). Differentiate S.typhi which is H2S negative.
XLD agar: red colonies with black centers.
DCA agar: colorless with black centers.
Enrichment media.
Biochemical testing:
Non lactose fermenter (nlf). (NB)
Motile. (NB)
Indole negative. (NB)
Mannitol: ferment. (NB)
Forms gas from glucose (S.typhi non gas-forming), ferments glucose.(NB)
H2S produced. S.typhi not.
Citrate positive. S.typhi citrate negative.
KCN: negative.
Dulcitol: positive. S.typhi non dulcitol fermenting.
Susceptibility testing: see S.typhi.
- Salmonella typhi
Clinical aspects:
S.typhi only a pathogen of man. Cause typhoid (do not confuse with rickettsial disease typhus),
diarrhoea, seticemia.
Specimens:
Stool, urine, blood culture.
Culture:
Blood agar: grey, smooth colonies.
Nutrient agar: colorless, smooth colonies.
SS agar: colorless. (very tiny black – H2S – cannot see it well. H2S negative.)
DCA agar: smaller, pale, smooth, shiny.
Enrichment media: selenite F, Rappaport media, Tetrathionate broth.
Non lactose fermenter.
Motile. (NB)
Indole negative. (NB)
Mannitol: ferment.
No gas from glucose (anaerogenic), ferment glucose. (NB)
H2S: little or none.
Citrate negative.
KCN: negative.
Dulcitol: not fermented. (Used to differentiate between Salmonella and Shigella. Has durham tube
for gas. Positive = gas + yellow color.)
Use the Kauffmann-White classification for serotyping of Salmonella.
The ID of serotypes depends on the detection of the O (somatic) and H (flagella) antigens by means
of agglutination with specific antisera.
O-antigens represent the side chains of sugar polymers projecting from the outer lipopolysaccharide
layer of the bacterial cell wall.
60 different types, heat stable, unaffected by heating at 1000C (water boiling point) for 2.5 hrs,
alcohol-stable, withstand 96% alcohol at 370C for 4 hrs. (this destroys the flagellar antigens.)
H-antigens represent determinant groups on the flagellar antigen.
Heat and alcohol labile – if 30 min at 100 0C it will detach the flagella from the bacteria = no
agglutination with H-antisera. Flagella antigens can be diphasic: phase 1 (specific), phase 2 (non-
specific).
Vi-antigen: S.typhi forms Vi antigen – covering the layer outside the cell wall.
Heat-labile, removed by heating 1 hr at 1000C.
O-groups A - Z : A= 2, or D= 9.
Groups A – E are human pathogens.
Eg. Kauffmann-White: S.typhi group A-; Oantigen 9, 12, Vi; H-antigen phase 1-d; phase 2-.
O, H and Vi antigens are positive with S.typhi.

Note: do not use colonies grown on MacConkey agar for typing.
Typing procedure:
Emulsify organism in saline. Use clean glass slide and place two drops on each end of slide.
Add poly O antisera to one drop and poly H to drop 2.
Look for agglutination. If positive in both = Salmonella.
Start typing with specific antisera.
Serology: Widal test.

Amoxacillin.
Ofloxacin.
Ceftriaxone.
Chloramphenicol. (NB)
● SHIGELLA
1. S.flexneri.
2. S.boydii.
3. S.sonnei.
4. S.dysenteriae.
Clinical aspects:
Bacillary dysentry (shigellosis), abdomonal pain, fever, watery stools, mucoid and blood, rarely
spreads from mucosa into blood.
Specimens:
Faeces, rectal swabs, rarely vomit.
NB: do a wet prep and look for numerous polymorphs (pus cells) and erythrocytes (blood).
Culture:
Aerobic or facultative anaerobic.
Blood agar: smooth, grey colonies.
Nutrient agar: smooth, colorless colonies.
MacConkey agar: nlf , but S.sonnei become pink with prolonged incubation – a late lactose
fermenter.
DCA agar – selective medium: pale small colonies.
SS agar: colorless colonies.
Selenite F broth – for growth and enrichment of S.flex. and S.sonnei.
S.flex & S.boydii S.sonnei S.dysenteriae
Motility non non non
Indole +/-- -- +
Mannitol + + --
Gas -- -- --
H2S -- -- --
Citrate -- -- --
KCN -- -- --
Dulcitol -- -- --
ONPG -- + --
Note: Klebsiella and Shigella are the only non-motile Enterobacteriaceae.
Shig.sonnei is a LLF.
Shig.dysenteriae very pathogenic. Dysentry is a notifiable disease.
Shigellas are differentiated by their somatic (O) antigens into serotypes identified by agglutination
with specific antisera.
Typing method:
Emulsify organism in a drop of saline.
Check first for auto-agglutination before adding 1 drop of specific antisera.
Mix or rotate 1 – 2 min. Look for agglutination.

Amoxacillin.
Ofloxacin.
Ceftriaxone.
Chloramphenicol.
Naladixic acid.
● SERRATIA
Looks exactly like Enterobacter on MacConkey. To differentiate:

Dnase: positive with Serratia.
Negative with Enterobacter.
Gas producer: Serratia – no.
Enterobacter – yes.
Serratia marcescens has bright red pigment.
● YERSINIA ENTEROCOLYTICA
Clinical aspects:
Enteritis, mesenteric lymphadenitis, infected animal bite, contamination of stored food.
(seems to be rarely encountered in humans in S.A.)
Hereditary haemochromatosis sufferers more susceptible.
(http://en.wikipedia.org/wiki/Yersinia_enterocolitica )
Specimens:
Blood, food, soil, faeces.
Gram negative cocco-bacilli; pleomorphic in older cultures.
Culture:
Aerobe.
Blood agar: non-haemolytic, smooth translucent colonies.
MacConkey agar: medium pin point colonies.
SS agar: nlf.
Motile at 220C (RT), non-motile at 370C (body temperature).
Indole: negative or positive.
Urease: positive.
Catalase: positive.
ONPG: positive.
Serotyping can be done with specific antisera for Yersinia enterocolytica.
Susceptibility testing: Chloramphenicol.

Tetracycline.
Usually rt penicillin, ampicillin and 3rd generation cephalosporins.
NON-FERMENTERS
● PSEUDOMONAS AERUGINOSA
Clinical aspects:
Natural habitat: environmental – water, soil, plants, hospital environment, pathogenic in swimming
pools, contact lens fluid, cosmetic solution, bowel coloniser, coloniser of upper respiratory tract,
UTI, ear, sinus, eye infections, endocarditis, septicaemia.
Specimens:
Urine, blood, pus swabs, sputum, burn swabs.
Culture:
Strict aerobe.
Temperature range: 50C – 420C. (Ps. aeroginosa 420C)
Blood agar: large colonies.
Pigment production: pyocyanin (blue-green, soluble in water and chloroform), fluorescin (yellow-
green, soluble in water; not chloroform), pyorubin (dark brown).
Distinctive musty smell.
Sheen in reflective light.
Oxidase positive.
Motile.
N2 gas produced.
Citrate positive.
KCN positive.
O/F test (Hugh Leiffson): oxidative.
Sellars media (stab plus slope innoculation): slant:green, but: blue or no change.
Susceptibility testing: Amikacin.
Gentamycin.
Ciprofloxacin.
Ofloxacin.
Ceftazidime.
● ACINETOBACTER ANITRATUS (= ACINETOBACTER BAUMANNII)
Clinical aspects:
Free living saprophyte in soil and water, regular contaminants in hospital environment, broncho-
pneumonia, septicaemia, skin and wound infections.
Specimens:
Blood cultures, pus swabs, burn swabs, endotracheal tube, urine.
Gram negative bacilli. On solid media – cocco-bacilli.
Culture:
Strict aerobe.
Blood agar: some can be haemolytic, white to cream smooth colonies.
MacConkey agar: non lactose fermenting colonies, slightly pink.
Oxidase negative or slow oxidase positive.
Non-motile.
No N2 gas produced.
Citrate: positive.
KCN: positive.
O/F test: oxidative.
Sellars media: slant: blue, butt: no change, yellow band with 10% dextrose.
Susceptibility testing: All strains are resistant to penicillin.

Hospital strains are rt Pg, ceph, E, T, C.
Use same sens as for gram negative bacilli, plus also report Ceftazidime.
ANAEROBES
ANAEROBIC BACILLI
● ACTINOMYCES spp
Clinical aspects:
Human actinomycosis – chronic granulomatous infection, lumpy jaw.
http://en.wikipedia.org/wiki/Actinomyces_israelii
Specimens:
Pus, pus swabs, blood culture.
Gram positive branching bacilli. Looks like fungal hyphae. Non-acid fast with Ziehl Neelsen.
Non endospore forming.
Culture:
Anaerobic or facultative anaerobic.
Responds to penicillin, amoxicillin.
● BACTEROIDES FRAGILIS
Clinical aspects:
Intra-abdominal infections, brain abscesses, oral infections, pleuro-pulmonary infections, pelvic
infections, bacteremia, endocarditis, skin and bone infections.
Specimens:
Pus swabs, throat swabs, cervical swabs, body fluids, blood cultures.
Gram negative pleomorphic bacilli.
Culture:
Anaerobic.
370C (body temperature) optimal.
Blood agar: convex white to grey, semi-opaque and glistening. Can be haemolytic.
Brucella agar: grey glistening colonies, foul smell.
Aesculin agar: hydrolyse (black).
Growth stimulated by bile.
Laboratory identification:
GNB (gram negative bacillus), pleomorphic.
Brucella agar with id 8-ring.
Aesculin agar: contains esculin and ferric citrate – hydrolysis (blackening around the colony).
Catalase positive.
Indole negative.
Susceptibility:
Use Wilson Chalgren media with metronidazole disc.
● CLOSTRIDIUM PERFRINGENS
http://en.wikipedia.org/wiki/Clostridium
Clinical aspects:
Gas gangrene, myonecrosis (necrosis of individual muscle fibers), food poisoning (C.botulinum and
C.perfringens).
http://en.wikipedia.org/wiki/Clostridium_perfringens
Specimens:
Wound and pus swabs, tissue, food samples, blood culture.
Gram positive bacilli.
Large with stubby or square ends.
Spores are formed only in absence of fermentable carbohydrates: subterminal and bulging.
Divided into saccharolytic and proteolytic clostridia on biochemical reactions.
Saccharolytic clostridia: grow rapidly in carbohydrate media with production of acid and abundant
gas; ferment glucose, cooked meat broth (Robertsons meat), but won't digest the meat; sour smell
and the meat is reddened, with acid and gas – eg. C.perfringins.
Proteolytic clostridia: digest meat, liquify gelatin, coagulate serum; cooked meat (Robertsons) –
blackening of the meat and the volume will reduce with formation of foul-smelling products – eg.
C.tetani.
Culture:
Anaerobic.
37 – 45 0C.
Blood agar: double zone of haemolysis (1st zone of complete haemolysis due to the theta toxin;
2nd wider zone of incomplete haemolysis due to alpha toxin).
Colonies are large, round, smooth, regular and slightly opaque.
Nagler plate (Willis and Hobbs media): selective media for clostridia. If neomycin is added it
makes media more selective for C.perfringens. The media is a lactose egg-yolk milk agar medium
with neomycin sulphate; the indicator is neutral red; the neomycin inhibits staphs and bacillus
species.
Several clostridia produce phospholipase enzymes (lecithinase) that will give rise to opalescence in
both human serum and egg yolk media. The Nagler reaction produced by C.perfringens is
neutralized by C.perfringens antitoxin.
Non-motile.
Catalase: negative.
Ferment glucose, lactose, sucrose, maltose, mannitol.
Dulcitol not fermented.
Gelatin: liquified.
H2S produced.
Milk media: stormy clot (acid clots the milk causing the protein to coagulate; the gas breaks up the
clot).
Indole negative.
Urease negative.
Susceptibility testing: Pg G preferred drug for gas gangrene.

Metronidazole.
Food poisoning:
C.perfringens type A produce heat resistant spores. Between 4 – 8 hrs after having eaten
contaminated food, develop acute abdominal pain and diarrhoea; nausea may occur but no
vomiting. Symptoms last for 12 – 18 hrs. Enterotoxin is synthesized during sporulation.
Types of contaminated foods: roasts, poultry, fish and stews. Also raw mwat at slaughter.
Clostridium germinates at cooking; multiply at cooling. 106 to 107 viable cells / gram of food will
cause symptoms.
ANAEROBIC STREPTOCOCCI
1. PEPTOSTREPTOCOCCUS spp
2. PEPTOSTREPTOCOCCUS ANAEROBIUS
3. PEPTOCOCCUS NIGER
Clinical aspects:
These species are normal flora in 1) the mouth, 2) urogenital tract and 3) gastrointestinal tract.
Human infections: 2nd most common encountered in human infections.
Very important in pleuropulmonary disease, brain abscesses, gynecological infections.
Specimens:
Pus swabs, cervical swabs, body fluids, blood cultures.
Gram positive cocci.
Culture:
Anaerobic.
Blood agar: small white colonies.
To identify – culture AER and ANAER.
Make a smear: gram pos. cocci.
Brucella agar with SPS (sodium polyanethol sulfonate).
Peptostreptococcus species: resistant to SPS (< 12 mm).
Peptostreptococcus anaeobius: sensitive to SPS (< 12 mm).
Peptococcus niger: black pigment.
Susceptibility testing: No need to test. Treatment of choice is Penicillin.
YEAST
● CANDIDA ALBICANS
Clinical aspects:
Oral candidiasis (thrush), cutaneous candidiasis (hands, feet, trunk), candida vaginitis, paronychia
(cuticle), respiratory tract, UTI, meningitis.
Specimens:
Sputum, skin, pus swabs, vaginal swabs, csf, biopsies, tissue, blood culture.
Gram positive yeast. C.albicans forms multicellular branched filaments from unicellular forms.
Culture:
Aerobe.
370C (body temperature) and RT.
Blood agar: raised, rounded white colonies; larger with prolonged incubation, dull appearrance.
Sabouraud Dextrose agar (contains dextrose, peptone, pH 5.4 – 5.6): white dull colonies. Incubate
day 1 at 370C, then RT.
Corn meal agar with Tween 80: inoculate with yeast, put coverslip on and incubate at 250C;
examine microscopically for chlamydospore formation.
Germ tube test:

Bovine or human serum – 9 drops. Emulsify yeast in serum.
Incubate at 370C for 1 – 2 hrs.
Look for germ tubes or pseudomycelium. If positive, id as C.albicans.
If negative, do further tests to id the yeast.
Treatment:
Nyastatin, Amphotericin B.
● CRYPTOCOCCUS NEOFORMANS
Clinical aspects:
Cryptococcosis, meningeal cryptococcosis, pulmonary cryptococcosus, cutaneous (skin) lesions,
HIV patients.
Specimens:
CSF, sputum, skin swabs, autopsy tissue, biopsies.
Yeast-like fungi. Heat fixation causes yeast to distort badly.
If numerous, can be seen on gram stain. They produce an extracellular polysaccharide capsule.
Do indian ink preparation: 1 drop of CSF onto a glass slide with diluted indian ink. Look for dark
yeast cells surrounded by a clear halo (refractile capsule).
Never forms a pseudomycelium.
Culture:
Aerobe.
220 (RT) and 370C (BT)(best at 37).
Blood agar: greyish slow growing colonies.
Sab dex agar: 1 at RT and 1 at BT: non descriptive colonies, slightly opaque; slime-producer like
klebsiella.
Potato dextrose agar: to enhance the growth and capsular formation.
Selective media: Tryptan blue: dark blue colonies at RT.
Niger seed agar: dark brown colonies at 370C (BT).
Urease: positive.
Treatment: Amphotericin B.
PART 2: MICROBIOLOGY: TESTS AND MEDIA :

http://www.scribd.com/doc/19540238/microbiologytests-and-media
NOTES FROM CAPE PENINSULA UNIVERSITY OF TECHNOLOGY (FMR. CAPE TECH.),
BIOMEDICAL TECHNOLOGY, 2003.
MORE COURSE NOTES AT: http://www.scribd.com/people/documents/2135965/folder/83622

Clinical Microbiology Bacteriology

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MEDICAL MICROBIOLOGY: BACTERIOLOGY

Susceptability testing: Penicillin.

Susceptibility testing: see notes for Staph. aureus.

Susceptibility testing: See notes for Staph. aureus.

● Lancefield group A, B and D.

Streps can be differentiated by: beta-haemolysis and alpha-haemolysis.

Streps microscopically: Gram positive cocci in chains.

Streptococcus pyogenes (Group A) (β−hemolytic)

Susceptibility testing: Penicillin.

Streptococcus agalactiae (Group B) (β−hemolytic)

Streptococcus (Enterococcus) faecalis (Group D) (variable hemolysis)

Susceptibility testing: Penicillin 8.

Streptococcus viridans (α -strep)

Streptococcus pneumoniae (Pneumococcus) (α−strep)

Susceptibility testing: Penicillin.

Neisseria gonorrhoea (Gonococci)

Susceptibility testing: Penicillin.

Neisseria meningitidis (Meningococcus)

Susceptibility testing: Penicillin.

Serological cross agglutination between N. meningitidis and N. lactamica.

Gram negative, oxidase positive diplococcus.

Susceptibility testing: Cotrimoxazole.

Susceptibility testing: Penicillin.

Susceptibility testing: Penicillin.

Susceptibility testing: Penicillin.

Susceptibility testing: INH.

Susceptibility testing: Cotrimoxazole.

Specimens: As for Hflu.

Microscopic examination: As Hflu.

Susceptibility testing: As for Hflu.

Susceptibility testing: Cotrimoxazole.

Antigenic structure: Serotyping done.

Susceptibility testing: as for E.coli.

Susceptibility testing: as for E.coli.

Susceptibility testing: same as for E.coli.

Susceptibility testing: see S.typhi.

O, H and Vi antigens are positive with S.typhi.

Susceptibility testing: Cotrimoxazole.

S.flex & S.boydii S.sonnei S.dysenteriae

Motility non non non

Susceptibility testing: Cotrimoxazole.

Looks exactly like Enterobacter on MacConkey. To differentiate:

Serratia marcescens has bright red pigment.

Susceptibility testing: Chloramphenicol.

● ACINETOBACTER ANITRATUS (= ACINETOBACTER BAUMANNII)

Susceptibility testing: All strains are resistant to penicillin.

Responds to penicillin, amoxicillin.

Susceptibility testing: Pg G preferred drug for gas gangrene.

Susceptibility testing: No need to test. Treatment of choice is Penicillin.

Germ tube test:

PART 2: MICROBIOLOGY: TESTS AND MEDIA :

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