Bacteriology

Bacteriology

I. Parts of a Bacteria
1. Capsule- slimy layer; colonies may appear mucoid; a bacterial structure which
resists phagocytosis; a virulence factor
o Composed of 98% water and 2% glycoprotein or polysaccharide
o Bacillus anthracis: capsules contain D-glutamate
o Neufeld-Quellung test:serelogical typing for capsules
o Encapsulated organisms: S. pneumoniae, N. meningitidis, Mycobacterium, H.
influenza, K. pneumoniae, B. anthracis
2. Cell wall- it gives shape to the bacteria; aka peptidofgycan layer basis of gram stain
Reagent Gram POSITIVE Gram NEGATIVE
PRIMARY STAIN Crystal Violet Violet Violet
MORDANT Gram’s Iodine Violet Violet
DECOLORIZER Gram’s alcohol (w/ Violet Colorless
(CRITICAL) acetone)
COUNTERSTAIN Safranin Violet Pink
Hucker’s modification: Crystal violet + 1% ammonium oxalate
o Gram positive- thick peptidoglycan layer; techoic acid (none in Gram negative)
Insoluble to alcohol
All cocci are gram-positive except: Neisseria, Veillonella, Moraxella
Actinomyces, Streptomyces, yeasts and molds are gram positive
o Gram negative- thin peptidoglycan; Lipopolysaccharide
Soluble to alcohol
All bacilli are gram-negative except: Mycobacterium, Clostridium,
Corynebacterium, Bacillus, Erysipelothrix, Lactobacillus, Listeria
All spiral organisms are Gram-negative
o Not GRAM-STAINED: Rickettsia, Chlamydia, Ureaplasma, Mycoplasma,
Spirochetes
3. Plasma membrane- site for energy synthesis; osmotic or permeability barrier;
regulates transport of nutrients in and out of the cell
4. Pili or fimbriae- serves as adherence factors (adhesins); virulence factor
Common pili- bacterial adherence
Sex pili- for gene transfer
Nucleic acid- Chromosome (for reproduction)
Plasmid- DNA which carries antibiotic-resistant genes
Endospores- structure resistant to heat (boiling), cold, drying and chemical agents;
contains calcium dipicolinic acid
Organisms: Bacillus (aerobic) and Clostridium (anaerobic)

Flagella
Monotrichous- flagellum at one polar end a.
Amphitrichous- flagella at two polar ends b.
Lopotrichous- tufts of flagella c.
Peritrichous- flagella surrounding the bacterial surface d.
o MOTILITY
Listeria monocytogenes- tumbling motility
Capnocytophaga- gliding motility
Campylobacter jejuni- darting motility
Spirochetes- corkscrew motility

| IF YOU WANT TO TOP. WAKE UP! 1

 Bacteriology

Kingella- twitching motility

Non-motile Shigella, Klebsiella, Y. pestis (enterobacteriaceae),
encapsulated organisms
II. Bacterial Requirement for Growth
1. Oxygen requirement
Obligate aerobe: Mycobacterium
Obligate anaerobe: Veillonella, Actinomyces
Facultative anaerobe: Most pathogenic bacteria
Microaerophiles: 21% O2; Campylobacter
Aerotoleranr anaerobes: Lactobacillus
Capnophilic: 5-10% CO2
2. Temperature
Psychrophilic: 0-10degreesCelsius
• Yersinia enterocolitica
• Listeria monocytogenes
Mesophilic: 20-45 degreesCelsius (Most pathogenic bacteria)
Thermophilic: 50-125 degreesCelsius (Thermus aquaticus)
3. pH
acidophilic
neutrophilic Most pathogenic bacteria
basophilic
4. Salt concentration- ”halophilic”
7.5% NaCl = Staphylococcus aureus
6.5% NaCl = Enterococcus faecalis
0%, 3%, 5%, 8%, 10% = Vibrio
5. Moisture
6. Nutritional requirement

III. Methods of Microbial Control

1. Disinfection & Sterilization: methods originated from as the concept of aseptic
surgery
2. Disinfection- process that eliminates a defined scope of microorganisms
Disinfectants: chemical agents applied to inanimate objects
• Halogen (chlorine, iodine, fluorine), heavy metals, aldehydes
• QUATS (quaternary ammonium compounds) i.e. benzalkonium
chloride and zephiram; easily inactivated by organic compounds
-Pseudomonas aeruginosa grown in ammonium acetate agar are
resistant to QUATS
• Best disinfectant: 10% sodium hypochlorite (household bleach)
Antiseptics: substance applied to the skin for the purpose of eliminating or
reducing the number of bacteria present
• alcohol
• Tincture of iodine- iodine + alcohol
• Iodophor- iodine + detergent (or QUATS)
• Chlorhexidine- antiseptic of choice if patient is allergic to betadine

3. Sterilization - refers to destruction of all forms of life including SPORES

A. Sterilization Methods: Physical Methods

| IF YOU WANT TO TOP. WAKE UP! 2

 Bacteriology

a. Moist Heat- sterilization method of choice for heat-stable objects

Autoclave (Steam under pressure)
o To sterilize used and unused media; 121degreesCelsius, 15mins, 15psi
o Indicator: Bacillus stearothermophilus
Tyndallization (Flowing steam) / Arnold sterilizer
o 100degCelsius for 30mins for n3 consecutive days
Inspissation
o To sterilize media with protein
o 75-80degCelsius for 2hours for 3 consecutive days

b. Dry Heat- commonly used to sterilize glassware

Oven - 160-180degCelsius for 1-2hours
- Indicator: Bacillus subtilis var niger
Incineration- heating in very high temperature to dispose infectious wastes
Flaming - to sterilize loops and needles
Cremation - burning materials to ashes to control diseases

c. Filtration- sterilization methods for vaccines and antibiotics; accomplished with the
use of high-efficiency particulate air (HEPA) filters (0.3um)
Millipore- gives 100% sterility; pore diameter: 0.22um

B. Sterilization Methods: Chemical Methods

a. Ethylene oxide- most commonly used chemical sterilant;
• Indicator: Bacillus subtilis var globiji
b. Formaledhyde/ hydrogen peroxide- to sterilize HEPA filters
c. Glutaraldehyde
d. Peracetic acid

C. Disinfection
a. Physical Method
• Pasteurization (63degCelsius for 30mins)- eliminates food-borne pathogens
and organisms responsible for food spoilage.
• Boiling (100° C)- kills most microorganisms in approximately 10 minutes

b. Chemical Method
• Antiseptics
• Disinfectant

IV. Specimen Collection and Processing

1. Swab (aerobic collection)
Cotton- toxic to Neisseria; good for virus
Calcium alginate- toxic for virus; good for Neisseria
Dacron & rayon swab- good for virus and Neisseria
2. Needle aspiration
3. Catheterization

o Specimen transport- samples are ideally transported within 2hours from the
time of collection
o Specimen preservation

| IF YOU WANT TO TOP. WAKE UP! 3

 Bacteriology

Boric acid- can maintain the colony count for urine culture
0.025% Sodium polyanethol sulfonate- anticoagulant of choice for
blood culture
Inactivates complement
Prevents phagocytosis
EDTA and Citrate- never used for blood culture
Heparin- can be used for viral culture
o Specimen storage
CSF- room temperature
Urine, stool, viral specimens, sputum swab- refrigerator temperature
Serum for Serology- -20degCelsius for 1week
Tissues/specimen for long-term storage kept frozen at -70degCelsius
V. Specimen for Bacteriology
1. Blood- collect before height of fever
Anticoagulant: 0.025% sodium polyanethol sulfonate
• Inhibits phagocytosis and inactivates complement
• Blood to anticoagulant ratio: 1:10
• Blood volume for children: 1-5mL
• Blood volume for adult: 10mL; 5mL aerobic; 5mL anaerobic
• To counteract: 1% gelatin
• It neutralizes bactericidal effects of the serum
• Turnaround time: 5days
2. Urine
Specimen of choice: Midstream clean-catch urine
Major cause of UTI: E.coli
Causes UTI in young women: S. saprophyticus
Colony count : MAC and BAP
• Colony count= # of colonies * loop factor
• *1000 (0.001mL); *100 (0.01mL); *10 (0.1mL)
• >100,000 CFU/mL- significant for UTI
• <10,000 CFU/mL- insignificant for UTI
3. Sputum
Can be contaminated by normal flora; the quality of the specimen must be evaluated
using Bartlett’s criteria; accept only when >10 PMNs <25 EC/LPF
Gram stain is performed to evaluate the quality of the sample
Collected ideally in the morning
Sputum decontamination reagents are used to break down mucous components of
sputum and to allow slower growing mycobacteria to grow.
• NALC (Na-acetyl-L-cysteine)-NaOH –gold standard
• NALC- digestant/mucolytic
• 2-4% NaOH- decontamination
• Oxalic acid- used for samples contaminated with P. aeruginosa
• Media: Lowenstein-Jensen, Middlebrook 7H10 and 7H11
4. Cerebrospinal Fluid
Collected via lumbar/spinal tap; collected in 3-4 tubes, tube #__ is used for gram stain
and culture
Storage: RT or 37degCelsius for no longer than 1hour—NEVER REFRIGERATE
Common organisms:
• G(-) bacillus which grows CHOC but not on MAC, BAP=H. influenzae
| IF YOU WANT TO TOP. WAKE UP! 4
 Bacteriology

• G(-) diplococci which grow on CHOC but not on MAC, BAP=N. gonorrhoeae
5. Throat and Nasopharyngeal Swab
Most abundant normal flora-Viridans group
Most common pathogen-S. pyogenes
Nasopharyngeal swab is the specimen of choice to detect carrier state of N. meningitidis,
B. pertussis, and H. influenza
6. Stool
Detection of enteric stool pathogen
Stool specimen must be placed in a transport medium if cannot be processed within
2hours of collection. CARY-BLAIR Medium
Routinely screened for Salmonella, Shigella, Campylobacter
Media: Eosin-Methylene Blue Agar, Salmonella-Shigella Agar, Hektoen Enteric Agar,
MacConkey Agar
7. Vaginal/ Urethral Swab
For diagnosis of postpartum endometriosis
Transport medium: Todd-Hewitt Medium
VI. Methods of Bacterial Identification
1. Staining Characteristics
2. Culture
3. Antimicrobial Susceptibility Testing
4. Biochemical tests

Staining Characteristics

Purpose: to observe the appearance of the bacteria ; to differentiate organisms; to

reveal chemical structure
Staining Techniques
Simple stain- the color of the stain is the resultant color
Indirect/Negative- i.e. India ink, Borris, Nigrosin stains the background
Special Stain- to help visualize and identify structures and substances in sections
Capsular stains- Muir, Anthony’s, Tyler, Hiss
Spore stains- Dorner’s, Schaeffer-Fulton, Wirtz and Conklin
Flagellar stains- Leifson, Grays, Fisher and Conn
Metachromatic granules-Albert’s, Neisser, Ponder, Methylene blue, Lindergran
Nucleic acid Feulgen
Polar bodies Wayson
Spirochetes Levaditi, Warthin-Starry, Fontana-Tribondeau
Rickettsia Machiavello
Differential Staining- uses more than one dye; used to differentiate organism
o GRAM STAIN and ACID FAST STAIN

ACID FAST STAIN

o AFO- refer organisms which are difficult to stained but when stained, they are
difficult to decolorize due to the presence of mycolic acid / hydroxymethoxy acid.
o Acid Fast Organisms: Mycobacterium, Nocardia (partial acid fast).
Cryptosporidium (Modified Acid Fast)

Ziehl-Neelsen/Hot method Kinyoun/Cold Method

(best method) (for tissues)
Primary Stain Carbolfuchsin Carbolfuchsin
Mordant Heat Heat
| IF YOU WANT TO TOP. WAKE UP! 5
 Bacteriology

Decolorizer Acid alcohol Acid alcohol

Counterstain Methylene blue/ Malachite green Methylene blue/ Malachite green
ACID FAST ORGANISMS- stains RED
NON- ACID FAST ORGANISMS-stains BLUE
Pappenheim’s- to differentiate M. smegmatis blue from M. tuberculosis red
Baumgarten’s- to differentiate M. leprae red from M. tuberculosis blue
Fite-Faraco- uses HEMATOXYLIN
MODIFIED ACID FAST STAIN- Cryptosporidium & Cyclospora

Culture
Purpose- contains nutritional components needed for bacterial growth
Types of culture
o Pure culture- one organism
Streak plate (best), pour plate, selective medium
o Mixed culture- more than two organisms
o Stock culture- American type culture selection

Classification of Culture Media

I. According to consistency
a. Liquid- no solidifying media i.e.no solidifying agent (APW, BHI agar)
b. Semi-solid- contains 0.5%-1% agar i.e. SIM, LIM
c. Solid media- 2-3% agar
d. Biphasic media- both solid and liquid media
II. According to use
a. General purpose media- contains the only necessary nutrition to support
bacteria growth, for routine cultivation of bacteria.nutrient agar
b. Enrichment media- for enhanced growth of organisms.selenite tetrathionite
c. Enriched media- for fastidious organisms.CAP and BAP
d. Selective media- promotes growth of a certain organism while inhibiting the
growth of others
i. Inhibitors for gram(+) organism-crystal violet and sodium
desoxycholate
ii. Inhibitors for gram(-) organism-sodium azide, potassium tellurite
iii. Lowenstein-Jensen – malachite green as inhibitor
iv. CAP with antibiotics- Neisseria
v. CTBA- C. diphtheriae
vi. CIN- Y. enterocolitica
vii. McBride- L. monocytogenes
viii. Castaneda- Brucella
ix. Petragnani- M. tuberculosis
x. SSA- Salmonella, Shigella
xi. BSA- Salmonella
xii. TCBS-Vibrio
xiii. BCYE- Legionella
xiv. Thayer-Martin-Vancomycin, Colistin, Nystatin
xv. Modified Thayer-Martin- Vancomycin, Colistin, Nystatin,
Trimethoprim
xvi. Martin-Lewis- Vancomycin, Colistin, Anistomycin, Trimethoprim
xvii. New York City- Vancomycin, Colistin, Amphotericin, Trimethoprim
xviii. PLET- Polymyxin EDTA Thallous acetate
xix. CCFA- C. difficile
xx. Cetrimide agar- P. aeruginosa
xxi. Regan-Lowe- Bordetella
xxii. Modified Skirrow’s Media- Campylobacter
xxiii. CAMPY-CVA- Cefoperazone, Vancomycin, Azithromycin
xxiv. BCGA- F. tularensis
xxv. Fletcher’s- Leptospira
| IF YOU WANT TO TOP. WAKE UP! 6
 Bacteriology

xxvi. Ellinghausen McCullough Johnson Harris (EMJH)

xxvii. Kelly’s- Spirochetes
xxviii. McCoy’s cell- Chlamydia
xxix. Chick embryo- Rickettsia
xxx. Egg Yolk Agar- Lecithinase Test/Nagler’s Test

Medium Inhibitors Indicator Carbohydrate Fermenter Non-Fermenter

Crystal violet
MAC Neutral red lactose Red/pink colorless
Bile salt
DCA Bile salt Neutral red lactose Red/pink colorless
Bile salt
SSA Neutral red lactose Red/pink colorless
Brilliant green
Salicin Green
Bromthymol
HEA Bile salt Lactose Yellow
blue
Sucrose w/black:Salmonella
Bromthymol
TCBS Bile salt Sucrose Yellow Green
blue
BSA Brilliant green Bismuth sulfite Glucose Black colonies: Salmonella
Eosin Y
Eosin Y
EMB Methylene Lactose Pink/purple Colorless
Methylene blue
blue
Xylose
XLD Bile salt Phenol red Lactose Yellow Red
Sucrose

e. Differential Media- to differentiate organism form one another

i. MAC- Lactose
ii. MSA-Mannitol
iii. BAP-Blood (Hemolytic pattern)
iv. TCBS-Vibrio
f. Transport Media
i. Stuart and Amies
ii. Cary-blair
iii. Transgrow
iv. Todd-hewitt

Antimicrobial Susceptibility Testing

Purpose- to determine the ability of antibiotics to inhibit bacteria, in vitro
Kirby-Bauer Method- the most common method used in AST
o Agar: Kirby Bauer
o pH: 7.2-7.4
o depth: 4mm (15-20mL)
o 150mm diameter- <12discs
o 100mm diameter- <5discs
o Distance of disk from center 24mm, between 2 disks 15mm
o Inoculum standardization: 0.5 McFarland Standard
99.5mL 1% H2SO4 + 0.5mL 1.175% Barium chloride
o SOURCES OF ERROR
Too light inoculum false susceptible
Too heavy inoculum false resistant
Depth of agar: too thin false susceptible too thick false resistant
Moisture on agar false resistant
Very dry agar false susceptible
Improper storage of disk: Indicator methicillin and penicillin

Biochemical tests

| IF YOU WANT TO TOP. WAKE UP! 7

 Bacteriology

1. Catalase test- enzyme in bacteria which breaks down H202 to H20 and 02
Differerentiates Staphylococcus and Micrococcus from Streptococcus
Positive: Bubble formation
Catalase rxn: bubble formation

2. Coagulase Test- causes bacterial cell to agglutinate in plasma

Indicator/reagent: rabbit plasma
Slide coagulase- detects bound coagulase
Tube coagulase- detects free coagulase

3. DNAse Test- deoxyribonuclease

DNA + methyl green = colorless
DNA + toluidine blue= pink
HCl precipitation method= clearing/halo around colonies
Positive organisms: Serratia, Moraxella, Staphylococcus
4. Modified oxidase test – differentiates Staphylococcus vs Micrococcus
Reagent- tetramethyl-p-phenylenediamine dihydrochloride in
dimethylsulfoxide (DMSO)
Positive: purple

5. CAMP test (Christie, Atkins, Munch, Petersen)

Differentitates Group B from Group A Streptococcus
Positive: enhanced hemolysis arrowhead hemolysis
Streptococcus agalactiae (+) with Staphylococcus aureus
Listeria monocytogenes block-type hemolysis with Rhodococcus equi
Arcanobacterium haemolyticum reverse CAMP

6. Hippurate Hydrolysis test- detects hydrolysis of sodium hippurate to benzoic

acid ferric chloride and glycine ninhydrin
Positive result: purple
Organisms- Campylobacter and S. agalactiae

7. Bile esculin test

Media: 40% bile
Reagent or indicator: ferric chloride
Positive result: blackening of agar

8. PYR test
Detects L-pyrrolidonyl arylamidase
A colony is placed on a filter paper with the substrate pyrrolidonyl-alpha-
naphthylamide (PYR)
Positive result: red color
Positive: S. pyogenes and Enterococcus

9. Staph A Coagglutination Test

Staphylococcus aureus (cowan strain) with protein A as inert partcles to
which Fc binds
Detects: S. pneumoniae, N. meningitidis, N. gonorrhoeae

10. Optochin test (contains ethylhydrocupreine hydrochloride)

For identification of Streptococcus pneumoniae

11. Bile solubility test

Blood agar plate with 10% sodium deoxycholate
Positive- Streptococcus pneumoniae
Negative- Viridans streptococcus / E. faecalis

| IF YOU WANT TO TOP. WAKE UP! 8

 Bacteriology

12. Superoxol test

Reagent:30% H2O2
Positive: bubble formation

13. Carbohydrate Fermentation Test

Media: cysteine trypticase agar
Fermentation: yelllow

14. Niacin test

Principle: Niacin ribonucleotide + aniline dye + cyanogen (toxic
substance)
Positive: bromide yellow-- M. tuberculosis (yellow); M. bovis (negative)

15. Arylsulfatase Test

used for identification of Mycobacteia rapid growers
reagent: tripotassium phenolphthalein disulfide
positive: M. fortuitum-chelonae (pink-red)

| IF YOU WANT TO TOP. WAKE UP! 9

 Bacteriology

16. Nagler’s Test/ Lecithinase test

Positive reaction: positive reaction is due to the alpha lecithinase C and
phospholipase C
Media: Egg yolk Agar
Positive” opaque colonies

17. Ascoli Test

Serological test for Bacillus anthracis

18. Quellung Reaction

The Quellung reaction is the gold standard method for pneumococcal
capsular serotyping.
The method involves testing a pneumococcal cell suspension with pooled
and specific antisera directed against the capsular polysaccharide.
Positive: Swelling of cells

19. Elek Test

It assesses toxigenicity of diphtheria toxin
Positive: lines of precipitation

20. Guthrie test

a screening for phenylketonuria (PKU) used to detect the abnormal presen
ce of phenylalanine metabolites in the blood.
small amount of blood is obtained and placed in a medium with a strain of
Bacillus subtilis

21. Nitrate reduction test

Reagent: sulfanilic acid and alpha-naphthylamine
Positive: red

22. ONPG Test

Purpose: determine ability of organism to produce beta galactosidase
Reaction: beta galactosidase converts ONPG to orthonitrophenol
Reagent: O-nitrophenyl-beta-D-galactopyranoside
Positive: yellow
Lactose fermenters!
Rapid Lactose Fermenters (KEE)
o Escherichia, Klebsiella, Enterobacter
Late Lactose Fermenters (SHY)
o Serratia, Hafnia, Yersinia
Non-Lactose Fermenters (PPM SEES)
o Proteus, Providencia, Morganella, Salmonella, Erwinia,
Edwardsiella, Shigella

23. IMViC
Indole test
Detects tryptopanase
Rgt: KOVACS reagent/PDAB
Positive result: Pink/Red ring
Methyl Red
Detects acid pH which indicates glucose fermentation
(+) E. coli; (-) . cloacae
Voges-Proskauer Test
Detects production of acetylmethyl carbinol (acetoin)
Reagent: alpha-naphthol and 40% KOH
Positive result: pink to red color
Positive: KESH—Klebsiella, Enterobacter, Serratia, Hafnia

| IF YOU WANT TO TOP. WAKE UP! 10

 Bacteriology

Citrate
Detects citrate utilization
Agar: Simmon’s citrate agar
Positive result: blue
24. Decarboxylase Test
Decarboxylase tests determine whether the bacterial species possess
enzymes capable of decarboxylating (removing the carboxyl group, COOH)
specific amino acids in the test medium
Indicator: bromcresol purple; (+) purple
E. agglomerans: triple decarboxylase negative

25. Acetate Utilization Test

ACETATE alkaline pH (blue)=positive / acid pH (r\green) =negative
(+) E. coli; (-) S. flexneri

26. Urea hydrolysis test (Urease test)

Agar: Christensen’s urea agar, Stuart urea broth
Positive: pink

27. Acetamide utilization test

Positive: blue
(+) P. aeruginosa; (-) S. maltophila

28. Malonate utilization test

Indicator: bromthymol blue
Positive: blue
Klebsiella—Enterobacter—Citrobacter—Hafnia: positive

29. Phenylalanine Deaminase (PAD)

Positive: green (Providencia, Proteus, Morganella)
(+)P. vulgaris; (-) E. coli

30. KCN broth test

Positive: Turbid
Klebsiella—Enterobacter—Proteus—Providencia

31. String test

For ID of Vibrio cholera
Reagent: 0.5% deoxycholate
(+) string like

32. MUG tests (UV light)

Reagent: 4-methylumbelliferyl-beta-D-glucuronide
(+) electric blue fluorescence: E. coli; (-) P. aeruginosa

33. Gelatin hydrolysis test

(+) gelatin liquefies: P. vulgaris; (-) E. aerogenes

SUMMARY OF BIOCHEMICAL ANALYSIS

| IF YOU WANT TO TOP. WAKE UP! 11

 Bacteriology

GRAM – POSITIVE COCCI • Pharyngitis / tonsillitis

• Erysipelas (St. Anthony’s fire)
1. Staphylococcus aureus • Scarlet fever
• “Buttery-looking” colonies on ß-hemolytic • Necrotizing fasciitis
• “jet black colonies on tellurite agar • Acute glomerulonephritis
TESTS • Rheumatic fever
• Catalase (+) Other Tests
• Coagulase (+) • Dick test Scarlet fever (+)
• DNAse (+) • Schultz-Charlton/Blanche Phenomenon
• VP/Nitrate (+)
• Mannitol fermenter (yellow colonies on 2. Streptococcus agalactiae (Group B Strep)
MSA) • Gram (+), ß-hemolytic
• Lysostaphin & Novobiocin sensitive • CAMP (+) with S. aureus
• Polymixin resistant • Hippurate hydrolysis (+)

Pathogenesis Pathogenesis
• TSS Toxin mediated • Neonatal meningitis
• SSS/Reiter’s DSE • Postpartum endometriosis

2. Staphylococcus epidermidis 3. Group C Streptococcus

• Catalase (+) , Y- hemolysis - Streptococcus equisimilis, Streptococcus equi,
• Novobiocin (s) Streptococcus dysgalactiae, streptococcus
zooepidermis
Pathogenesis
• Prosthetic heart valve infection • Gram (+), ß-hemolytic
• Taxo resistant
3. Staphyloccocus saprophyticus • SXT (S)
• Catalase (+) , Y- hemolytic
• Novobiocin (R) Pathogenesis
• Bacteremia
Pathogenesis • Pneumonia
• Most common cause of UTI in young • Cellulitis
women • Abscess

4. Staphylococcus intermedius 4. Group D Streptococci (non 0- enterococci)

• Slide coagulase (+) - Streptococcus bovis and Streptococcus equinus
• PYR(+) • 6.5 % NaCl (S)
• Mannitol(-) • Penicillin (S)
• Vancomycin (resistant)
5. Staphylococcus lugdunensis • PYR (-)
• Slide coagulase (+) • Bile esculin (+)
• Mannitol (+)
• Narrow ß-hemolysis Pathogenesis
• PYR (+) • Colon cancer (S. bovis)
• Endocarditis
• Septicemia
Micrococci (OXIDIZER)
• Y-hemolysis on BAP Enterococcus
• Modified oxidase (+) • 6.5% NaCl (+) growth
• Catalase (+) • Penicilin & Cephalosporin (resistant)
• Gram (+) • Taxo A (resistant)
• Lysostaphin • Bile esculin (+),PYR (+)
• Taxoa (S)
• Furazolidone ® 1. Enterococcus faecalis
• Pyruvate (+)
Streptococcus • Arabinose (-)
1. Streptococcus pyogenes (Group A strep) • Sorbitol (+)
• Gram (+) , ß-hemolytic
• TAXO A (0.02 – 0.04 units) (S) 2. Enterococcus faecium
• PYR (+) • Pyruvate (+)
• Catalase (-) • Arabinose (+)
• SXT (resistant) • Sorbitol (-/+)
• Group A Streptococcus 3. Streptococcus pneumoniae
Pathogenesis • Diplococcus pneumonia/pneumococci

| IF YOU WANT TO TOP. WAKE UP! 12

 Bacteriology

• Capnophilic alpha- hemolytic • “wagon wheel”

• “Dome-shaped” colonies
• “nailhead colonies or “crater-like or GRAM POSITIVE SPORE FORMING BACILLI
“checker” BACILLUS
Tests
• Bile solubility (+) 1. Bacillus anthracis
• Taxo P (S) • “Anthrax bacillus/bamboo fishing rod”
• Neufeld – Quellung (+) • Only with “D-glutamate capsule”
• Inulin fermenter • “box-car morphology (henry’s)
Other tests • “gamma hemolytic and non-motile”
• Francis Test (+) pneumonia • Beaten egg white appearance”
• “Medusa head colonies/Lion head/cut-
Pathogenesis glass appearance”
• Lobar pneumonia • “string of pearl”
• Otitis media #1 • In gelatin “inverted fir tree/inverted pine
• Bacterial meningitis tree”

4. Viridans TESTS
• Gram (+), alpha- hemolytic • Lecithinase (+)
• Optochin (resistant) • Ascoli test (+) for anthrax
• Bile solubility
• Taxo A (resistant) Pathogenesis
• SXT (S) • Cutaneous anthrax (malignant
pustule/black eschar)
5. Abiotrophia • Pulmonary anthrax (ragpicker’s,
• Thiol-requiring, pyridoxal –requiring. woolsorter’s, hide porter’s disease)
Satelliting strep • Gastrointestinal anthrax
• Requires vitamin B6
Others
• Glucose, sucrose, maltose fermentation
GRAM NEGATIVE COCCI • Pen G (S)
Neisseria • Neufeld –quellung (+)
• Catalase (+)
• Non-motile 2. Bacillus cereus
• Aerobic • “Fried rice bacillus”
• Capnophilic • ß-hemolytic and motile’

1. Neisseria gonorrhoeae TESTS

• “kidney, coffee bean shaped” intracellular • Salicin fermentation (+)
with in PMN • Growth at 45⁰C (+)
• Glucose fermenter • Glucose, maltose, sucrose (+)
• Superoxol (+) • Gelatin and PEA (+)
• PEN G (resistant)
Pathogenesis
• STD 3. Bacillus subtilis
• Opthalmia neonatorium • “Hay bacillus”
• Fitz-Hugh-Curtid Syndrome • “ß-hemolytic and motile”
• Perihepatitis • Source of bacitracin

2. Nesseria meningitidis Pathogenesis

• “Bean-shaped diplococci • “eye infection in heroin addicts”
• Oxidase (+)
• Glucose &Maltose fermenter CLOSTRIDIUM
1. Clostridium perfringens
Pathogenesis • Clostridium welchii
• Bacterial meningitis • “Frankel’s bacillus/gas gangrene bacillus”
• Meningococcemia • Double zone hemolysis
• Waterhouse-Freiderichsen Syndrome • Reverse CAMP (+); Nagler reaction (+)
• Stormy fermentation of milk
3. Moraxella catarrhalis
• Catalase (-), Y- hemolytic Pathogenesis
• Asaccharolytic • Food poisoning
• Butyrate disk (+) • Gas gangrene/myonecrosis
• “Hockey Puck colonies • Necrotic enteritis
• Nitrate (+) 2. Clostridium tetani
• ß-hemolysis
| IF YOU WANT TO TOP. WAKE UP! 13
 Bacteriology

• “tackhead bacillus/tetanus 1. Corynebacterium diptheriae

bacillus/drumstick/lollipop/tennis racquet • “Kleb-loeffler’s bacillus
bacillus • “Club-shaped appearance/palisade/picket
• Terminal spores fences/Chinese characters”
• Narrow zone of hemolysis on BAP
Pathogenesis • “babes Ernst granules” aka volutin
• Tetanus • Loeffer’s and Pai’s coagulated egg serum-
• Lock saw enhances granulation and pleomorphism
• Neurotoxin • “black colonies with brown halo in Tinsdale
• Spastic paralysis medium”
• “Risus sardonicus”-distorted grin • Black colonies on CTBA”

3. Clostridium botulimum TESTS

• “Von Ermengen’s bacillus/canned good • Nitrate (+)
bacillus” • Catalase (+)
• alpha- hemolytic • Elek test for in vitro toxigenicity test
• Neurotoxin • Schick test (susceptibility test)

Pathogenesis Pathogenesis
• Homed canned good • Diphtheria
• Infant botulism • Bull’s neck appearance
• Floppy baby syndrome
• Strabismus- wandering Eye 2. Corynebacterium ulcerans
• Urease(+)
4. Clostridium difficile • Starch/glycogen HOH(+)
• “barnyard/Horse stable door”
• “Chartreuse fluorescence/yellow Pathogenesis
Fluorescence • Mastitis in cattle
• Motile
• Glucose(+) 3. Corynebacterium pseudotuberculosis
• Urease (+)
Pathogenesis • Nitrate reduction (+/-)
• Antibiotic-associated pseudomembranous
colitis (clindamycin) 4. Corynebacterium xerosis
• Nitrate reduction (+)
GRAM POSITIVE NON-SPORE FORMING BACILLI • CHO fermentation: Glucose, Sucrose and
Maltose
1. Listeria monocytogenes
• Narrow band of ß-hemolysis in McBride’s 5. Corynebacterium pseudodiptheriticum
media • “Hoffmann’s bacillus”
• “Inverted Christmas tree/ umbrella-like in • Urease (+)
SIM” • Nitrate reduction (+)
• CAMP (+)
• Anton test (+) organism inoculated in 6. Corynebacterium minutissimum
rabbit’s eye • “Coral red fluorescence” on wood’s lamp
• Motile at RT, NM at 37⁰C (tumbling)
• Esculin (+) Pathogenesis
• Erythrasma
Pathogenesis
• Meningitis in immunocompromised 7. Corynebacterium urealyticum
patients • Rapid urease (+)
• “cole slaw and soft cheese” infection • Catalase (+)

2. Erysipelothrix rhusiopathiae 8. Corynebacterium jeikeium

• Non-Motile • “JK bacillus”
• Catalase (-) • Glucose fermenter
• “bottle brush/test tube brush-like/pipe-
cleaner pattern” MYCOBACTERIA
• H2s (+) on TSI 1. Mycobacterium tuberculosis
• “KOCH’s bacilli/tubercle bacilli”
3. Lactobacillus acidophilus • Cauliflower-like growth
• “Doderlein bacilli” • Niacin (+)
• Grown in tomato juice agar • Nitrate (+)
• TCH Nagler reaction (+)
• Pyrizinamidase (+)
CORYNEBACTERIUM 2. Mycobacterium ulcerans
| IF YOU WANT TO TOP. WAKE UP! 14
 Bacteriology

• Buruli ulcers • Lactose (-) except S. sonnei

• K/A no gas on TSI
3. Mycobacterium leprae
• “Hansen’s bacillus” a. Shigella dysenteriae – “Shiga’s
• “Cigar packet” bacillus”
• Lepra cells (+)-Large macrophage with b. Shigella flexneri
foamy cytoplasm • Flexner’s bacilli/strong’s bacilli
• Lepromin test- Skin • Mannitol (+)
o Fernandez- reaction (skin) occurs to c. Shigella boydii
signal a positive result in Lepromin test • “Newcastle – Manchester
o Mitsuda- delayed reaction (5-6weeks) bacillus”
in lepromin test • Mannitol (+)
• Catalase(+)
4. Mycobacterium bovis – d. Shigella sonnei
• water- droplet colonies • “Duval’s bacillus”
• ONPG (+)
5. Mycobacterium kansasii – • Mannitol (+)
• “yellow/cross-barred bacilli” • ODC (+)
• Nitrate reduction (+) • Catalase (+)
• Heat stable catalase (+)
• Tween 80 HOH (+) 3. Citrobacter (IMVIC: - + - +)
• ONPG (+)
6. Mycobacterium marinum – “swimming a. Citrobacter freundii
pool/ fish tank granuloma (H2S(+) ODC (+))
b. Citrobacter koseri
7. Mycobacterium gordonae – “tap water (indole (+), ODC (+))
bacillus”
4. Edwardsiella (IMVIC: + + - - )
8. Mycobacterium xenopi – “Bird’s nest TSI: K/A , gas (+) , H2S (+)
appearance on cornmeal agar”
Kleb --- Enterobacter--Serratia -------Hafnia Group
9. Mycobacterium avium – intracellular VP (+) GAS (+)
“ Battey’s bacillus” 1. Klebsiella pneumoniae (- - + + )
• Friedlander’s bacillus”
10. Mycobacterium terrae – “raddish bacillus” • Neufeld – quelling (+)
• LDC (+)
11. Mycobacterium fortuitum • TSI A/A + gas
• Arylsulfatase (+) • VP (+)
• MAC (+) without crystal violet • String test (+)
• Nitrate reduction (+)
• Heat stable catalase (+) 2. Klebsiella oxytoca
• Iron uptake (+) • LDC (+)
• Pyrazinamidase (+) • VP (+)
• Indole (+)
12. Mycobacterium chelonae
• Arylsulfatase and Pyrazinamidase (+) 3. Klebsiella ozanae
• MAC (+) without crystal violet • LDC (+)
• Heat stable catalase (+) • VP (+)

GRAM NEGATIVE ENTERIC BACILLI 4. Serratia

Enterobacteriaceae • Dnase (+)
• Facultative anaerobe/aerobe • Lipase (+)
• Motile except: Shigella, Klebsiella, and Y. • Gelatinase (+)
pestis • Possess Prodigiosin (red
• Glucose (+) pigment)
• Gas (+) except Shigella
• Catalase (+) except S. dysenteriae 5. Enterobacter (IMViC- - + + )
• Motile
1. Escherichia (IMVic : ++ - - ) • ODC
• TSI A/A gas (+) • TSI : A/A gas , H2O
• EIEC (Montezuma’s revenge on Turista)
• EIEC (Shigella – like infection) 6.Salmonella (IMViC - + - - )
• EPEC (non-inclusive infinite diarrhea) • NLF
• EHEC/VTEC (HUS) • H2S (+) except S. paratyphi A
• EAEC (Pediatric diarrhea) • TSI: K/A + gas / LIA : K/K
2. Shigella PROTEUS – PROVIDENCIA – MORGANELLA
| IF YOU WANT TO TOP. WAKE UP! 15
 Bacteriology

• Rapid urease (+) • Onion bulb rot in plants (cystic fibrosis)

• Deaminase (+) • Foot rot in man
• Motile
• MR (+) 2. Haemophilis aegypticus
• LIA: K/A • “KOCH –week bacillus”
• Resembles H. influenza
1. Proteus mirabilis biotype II
• ODC (+) Pathogenesis
• Ampicillin, cephalosporin, • Pink eye
chloramphenicol (S) • Brazilian purpuric fever
• Source of OX-K
TESTS
2. Proteus vulgaris • Glucose (+)
• Indole (+) • X factor(+)
• Ampicillin, cephalosporin, • V factor (+)
chloramphenicol ®
3. Haemophilus ducreyi
PROVIDENCIA • “school of fish/railroad
• Deaminase and citrate (+) track/finger print”

MORGANELLA TESTS
• Urease (+) • X factor (+)
• Deaminase (+) • Glucose (+)

YERSINIA Pathogenesis
1. Yersinia pestis • “soft chancre”
• “Plague bacillus”/6th century dse/ • STD ulcerative dse
black death”
• Vector: Xenopsylla cheopis 4. Haemophilus hemolyticus
• “hammered copper glistening • ß-hemolytic
colonies on BAP • mistaken for S. pyogenes
• Best gown in CIN
• “stalactite growth on broth” 5. Haemophilus parainfluenzae
• Glucose (+)
Pathogenesis • Sucrose (+)
• Bubonic plague • V factor (+)
• Pneumonic plague • D-Aminolevulinic acid (+)
• Septicemic/Black death
PSEUDOMONADACEAE
2. Yersinia enterocolitica 1. Pseudomonas aeruginosa
• “safety pin appearance/bulls eye • Strict aerobe
on CIN • Motile
• Ornithine (+) • “overripe” grape/corn tortillas –
• Urease (+) like odor”
• ONPG (+) • Pyocyanin (blue pigment)
• Mannitol and sucrose (+) • Pyoverdin (green fluorescence)
TESTS
3. Yersinia pseudotuberculosis • ß- hemolytic
• OD (-) • Acetamide (+)
• Nitrate reduction (+)
HAEMOPHILUS • Oxidase (+)
• Capnophilic • Gelatin HOH (+)
• Oxidase (+) • Cetrimide agar (+)
• Catalase (+)
Pathogenesis
1. Haemophilus influenzae • Swimmer’s ear
• Pfeiffer’s bacillus • Jacuzzi / hot tub syndrome
• Mousy odor and dew drop • Erythema gangrenosum
colonies on media • Shanghai syndrome
• Y- hemolytic
TESTS BURKHOLDERIA
• Satellitism (+) 1. Burkholderia cepacia
• Neufeld – Quellung (+) Old names: Pasteurella kingae
• Glucose (+) • “dirt-like odor colonies

Pathogenesis TESTS
| IF YOU WANT TO TOP. WAKE UP! 16
 Bacteriology

• Lysine (+) • NON-MOTILE EXCEPT B. bronchiseptica

• Glucose (+) • Bordet-gengou media/potato blood glycerol
• Lactose (+) • Modified jones Kendrick charcoal
• Regan-lowe medium
Pathogenesis
• Onion bulb rot in plants (cystic fibrosis) 1. Bordetella pertussis
• Foot rot in man • “mercury droplet colonies”
• Oxidase (+)
2. Burkeholderia pseudomallei
• “Whitmore’s bacillus” Pathogenesis
“earthy odor on ashdown medium” • Whooping cough/pertussis

TESTS 2. Bordetella bronchiseptica

• Oxidase (+) • Rapid urease (+)
• Nitrate reduction (+) • Nitrate (+)
• Anginine (+) • Motility (+)
• Glucose (+) • Oxidase (+)
• Lactose (+) • Citrate (+)
• BAP(+)
Pathogenesis
• Malleidosis HELICOBACTER
• Vietnam time bomb
1. Helicobacter pylori
3. Burkholdera mallei old name: Campylobacter coli
• “glander’s bacillus • Urea (+)
Pathogenesis • Warthin – starry (+) for stain
• Glander’s disease • Skirrow’s agar
• Fancy
Pathogenesis
STENOTROPOMONAS • Peptic ulcer
• Large colonies with yellow pigment on TSA • Gastritis
and lavender green pigment • Gastric carcinoma

NON-FERMENTATIVE GRAM NEGATIVE BACILLI TESTS

• Catalase (+)
1. Acinetobacter baumanii • Cephalotin (S)
• “iraqibacter” • Nalidixic acid ®
• Catalase (+)
• Oxidase (-) CAMPYLOBACTER
• Non-motile
• “gummy colonies” • “S-shaped bacilli/seagull wings
• Fermenter • Darting motility
• Microaerophile
GRAM NEGATIVE BACILLI RECOVERED ON SPECIAL • Modified skirrow’s agar
MEDIA • Campy-BAP/Campy –CVA
• Modified charcoal cefoperazone
Brucella deoxycholate

• Non-motile 1. Campylobacter jejuni

• Catalase (+) • Growth at 42⁰C (+)
• Oxidase (+) • Hippurate HOH (+)
• Erythrol (+) • Nitrate reduction (+)
• Capnophilic • Cephalotin resistant
• Castaneda/Wisconsin agar • Nalidixic acid (S)

DISEASE: undulant/Gibraltar/Mediterranean fever Pathogenesis

• All require thanine except: B. abortus • Guillain-Barre syndrome
• All grow with basic fuschin except: B.suis
and B. canis FRANCISELLA

1. B. abortus (cattles) 1. Francisella tularensis

2. B. melliternsis (goats) • “Faint bipolar staining”
3. B. suis (pigs) • alpha-hemolytic
4. B. canis (dogs) • Multiply with Naegleria and Acanthamoeba
• Blood cystine glucose agar
BORDETELLA TESTS
| IF YOU WANT TO TOP. WAKE UP! 17
 Bacteriology

• Forshay test (S)

• Cystine and cysteine (+) 2. Plesiomonas shigelloides
• Motility (++)
Pathogenesis • Oxidase (+)
• Rabbit fever • LOA (+++)
• Tularemia
• Deerfly fever 3. Pasteurella multocida
• Water rat trapper disease • “musty odor”
• Shipping fever in cattle
LEGIONELLA
• Buffered charcoal yeast extract 4. Gardenerella vaginalis
• Bronchial washing Old name: Corynebacterium/ Haemophilus
vaginalis
1. Legionella pneumophila TESTS
• Pontiac fever • Clue cells on PAP smear
• Legionnaire’s disease • Whiff tests/ Shiff tests
• Broad street pneumonia
Pathogenesis
2. Legionella micdadei • Bacterial vaginosis
• Pitsburgh pneumonia
5. Chromobacterium violaceum
3. Legionella bozemanii • ß-hemolytic ammonium cyanide odor
• Wigas’s agent of pneumonia • violacein/violet pigment in DNAse agar

VIBRiONACEAE 6. Streptobacillus moniliformis

• Monotrichous • String of beads appearance
• Oxidase (+) rapid identification • Puff balls/puff ball appearance on broth
• Catalase (+) Pathogenesis
• Halophile • Rat bite fever and Spirillium monera
• TCBC (thiosulfate citrate bile salt sucose) • Hover hill fever
• Alkaline peptone water
HACEK
1.Vibrio cholerae • Subacute endocarditis
• String test (+) • Only catalase (+) A. actinomycetemcomitans
• Cholera red test (+) • Only indole (+) Cardiobacterium hominis
• “shooting star motility” • Only LDC (+) Eikenella corrodens
• Polymixin (classical (S) El tor ®
Pathogenesis 1. Haemophilus parainfluenzae
• Rice watery stool 2. Aggregatibacter actinomycetemcomitans
• Star-shaped colonies
Other tests Pathogenesis
• NaCl (+) • Periodontal disease
• Motility(+), Oxidase (+)
• LOA (+ + - ) 3. Cardiobacterium hominis
• Gelatin Liquefaction (+) • Rosette or flowerette arrangement
• 0129 (S) in G stain

OTHER GRAM NEGATIVE BACILLI 4. Eikenella corrodens

1. Aeromonas • Pitting on agar
• Oxidase (+) • Bleach-like odor
• Motility(+) Pathogenesis
• DNASE (+) • Clenched face and human bite
• 0129 (+) infection
• LOA (+ -+)
• Gelatin liquefaction 5. Kingella kingae – twitching motility

“You are who you want to be. WHO WILL YOU BE?”

-G’greens Odear (2019)

| IF YOU WANT TO TOP. WAKE UP! 18