Gram stain Procedure

Primary stain 10 seconds-

1 minute
Rinse
Mordant 10 seconds-
1 minute
Rinse
Decolorization
5-10 seconds
Rinse
Counter stain 10 seconds-
1 minute

Peptidoglycan in positive cell wall traps the Crystal violet, hence blue color
 Some Gram stains to
Gram positive cocci
remember! Visit the Gram
in pairs and chains stain Tutorial for more
Streptococcus organisms!
Gram negative rod – Fusiform
Shaped – Fusobacterium
species

Gram negative
bacillus
Resembling an
Enteric

Gram positive cocci in clusters,

Staphylococcus
Gram positive rod
most likely a
Bacillus species
Gram stain is used to assess quality of
Sputum specimen for culture
• Expectorated sputum specimen is rejected for bacterial
culture if judged to be “spit”:
– Perform Gram stain of sputum specimen
– If >= 25 epithelial cells /field
– Sputum is judged to be spit / not a sputum specimen
– Bacterial culture is not performed / it is rejected
– Request made for a new “deep cough” specimen
Bad Sputum Good
Sputum

10X objective 10X objective

 Most used Agar Media in Bacteriology
Blood agar- 5% sheep’s blood agar
Gauge hemolytic reaction of bacteria (alpha, beta, gamma)
Grow variety of Gram positive & Gram negative bacteria and
Yeast

Chocolate agar – “carmelized” blood agar – more enriched than

blood agar medium. Will support the growth of the same
organisms as blood agar Plus the more fastidious bacteria
like Haemophilus influenzae and Neisseria gonorrhoeae
 Most used agar media in Bacteriology
• MacConkey agar – Supports growth of Gram negative rods
(GNR) only (crystal violet inhibits Gram positive organisms)
Selective (only GNR grow ) and Differential (Lactose
fermentation can be determined)
Lactose fermentation = pink (neutral red indicator) (+)
non-lactose fermentation = no color (-)

• After media is plated incubate in aerobic and/or CO2

incubators at 35˚ C for 24 – 72 hours
 Newest Method for Organism
Identification
• MALDI-TOF mass spectrometry
– Matrix-assisted laser desorption/ionization
– Identification of organisms by analyzing proteins
Gram Positive Cocci

Staphylococcus
Streptococcus
Enterococcus
 Staphylococcus
Gram positive cocci inclusters
due to bound coagulase or “clumping factor”

All Staphylococcus are Positive for Catalase Enzyme

Staphylococcus aureus Coagulase Negative Staph

Coagulase Enzyme Positive Coagulase Enzyme Negative
+/- Yellow colony White colony
Beta hemolysis Most not hemolytic
 Catalase Enzyme Reaction
Negative Positive

Hydrogen Peroxide plus bacteria

Avoid blood agar when collecting Staph –

blood has innate catalase activity and will
give false positive catalase reaction
 Tube Coagulase Reaction

Rabbit plasma inoculated with organism

Incubate at 35˚C
Read at 4 hours and if negative read again at 24 hours

Negative tube coagulase =no clot

Coagulase negative Staph

Positive tube coagulase [at 4 or 24 hrs] =

Clot = Staph aureus
 Slide Agglutination Test to identify
Staphylococcus aureus

clumping

No clumping

• Clumping factor (coagulase) and protein A is found on the S. aureus cell wall.
• Fibrinogen and IgG to protein A is on the surface of the Staphaurex latex beads
• S. aureus will clump in the Staphaurex latex bead solution and
positively identifies Staphylococcus aureus.
• Coagulase negative Staphylococcus species remain milky and do not clump in
the solution.
•Advantage over tube coagulase – reaction time is 30 seconds vs. 24 hours
 Staphylococcus aureus
• Primary virulence factor:
– Protein A – surface protein, ability to bind immunoglobulin and
combat the immune response

• Diseases and associated toxin:

– Toxic shock syndrome (TSST-1 toxin)
– Scalded skin syndrome (Exfoliatin (SSS) toxin)
– Soft tissue infection (Panton valentine leucocidin toxin – PVL)
– Septic arthritis – primary cause
– Food poisoning / Enterotoxins – stable to
heating at 100*C for 30 minutes
– Bacteremia and endocarditis
• Toxins act as superantigens, recruit host
defense cells that liberate cytokines with
systemic effects
 Susceptibility issues –(1) MRSA
• Methicillin Resistance (MRSA =Methicillin resistant S. aureus)
– Altered Penicillin binding protein (PBP2) produced by the mecA gene
codes for resistance to oxacillin/methicillin/nafcillin
– All MRSA are considered resistant to semisythetic penicillins and
cephalosporins
– Vancomycin is drug of choice – although reduced susceptibility
reported
– Cefoxitin susceptibility testing is considered a more sensitive indicator
of resistance and provides better detection of MRSA than oxacillin
testing. Shown is a Kirby Bauer (KB) disk test that is resistant to
cefoxitin.
Staphylococcus aureus (MRSA)
Hospital Epidemiology Issue:
Nasal surveillance for MRSA
Nares is a colonization site for MRSA and the
most common surveillance site cultured.

ChromAgar is commonly used to detect MRSA.

It is a selective and differential media with
chromogenic substrates that turn specific colors
to ID Staph aureus. Cefoxitin is added to the
medium to select for methicillin resistant Staph
aureus (MRSA).

Molecular assays (MA) can also be used to

screen nares for MRSA. MAs increase the
sensitivity of detection (@5-10%) but increases
laboratory costs.

Mupiricin can be used to eliminate carriage

 Coagulase negative Staph (CNS)
@ 15 species infect humans

• Staph epidermidis – is the most common White non-hemolytic

colony
– Major aerobic component of normal skin flora
– Common cause of subacute bacterial endocarditis
– Pathogenicity from cell adhesion factors forming
biofilm on biologics and plastic
(Catheters and replacement joints) – can be
difficult to treat
• Staph saprophyticus –
– Urinary tract infections in the child bearing age female,
– This species of CNS adheres in greater #’s to epithelial cells
– Novobiocin resistant by KB disk test can be used as test to identify
Staph saprophyticus

resistant
 CNS & Related Cocci
• Staph hemolyticus –
– Can cause line related sepsis
– This CNS is hemolytic on blood agar
– Do not confuse with Staph aureus
• Staph lugdunensis
– Positive PYR test Pos Neg
– Perhaps greater pathogenicity than other species
• Micrococcus species
– Mustard yellow colored colony
– Catalase positive / Gram positive cocci in tetrads
– Tube coagulase negative
– Does not ferment glucose
• Staphylococcus ferment glucose
– Environmental contaminate
 Streptococcus

Gram positive cocci in chains and pairs

Catalase enzyme negative
Grouped by hemolytic pattern on 5% sheep’s blood agar
– Alpha – greening of agar, partial hemolysis of RBCs
• Viridans Streptococcus, Strep pneumoniae,
Granulicatella/Abiotrophia
– Beta – clearing of agar, complete hemolysis of RBCs
• Beta hemolytic Streptococcus groups A - G
– Gamma – no clearing of agar, intact RBCs
• Streptococcus bovis (gallolyticus)
 Beta hemolytic
Streptococcus
• Beta Streptococcus are grouped using the “C” (CHO)
carbohydrate in the cell wall of the bacteria
• Identifies the Beta Strep groups – A, B, C, F, and G that are
most common cause of human infections
• Lancefield grouping system uses the “C” CHO in the cell
wall of Streptococcus (antigen) in a slide agglutination
reaction with purchased monoclonal antibody attached to
latex beads
See below slide for a positive and negative reaction.
• Immunologic typing is more accurate than biochemical
testing to group Streptococcus +
-
 Streptococcus pyogenes
• Genus/species for Group A beta Streptococcus [GAS]
Biochemical identification:

– Bacitracin KB sensitivity test – inhibited, no growth @ disk

– This test is not specific for Group A , it x-reacts with group C
– “A” disk is the Bacitracin disk

– PYR (pyrrolidonyl arylmidase) reaction

• Organism spotted on moist disk
• 2 min – RT incubation
• Add Cinnamaldehyde reagent
• Look for color reaction
• Pink = positive = Strep pyogenes
• This test is not exclusive for Strep pyogenes – Enterococcus and
Staph lugdunensis are also positive for PYR

– Therapy : Penicillin or Cephalosporin antibiotics

 Primary virulence factors :

M Protein – prevents
phagocytosis

Capsule – hyaluronic capsule

protects from phagocytosis

– Streptolysin O and Streptolysin S toxins

• Comprise the ASO titer dtermination
• Cell toxins lead to evasion from the immune system
• O toxin is oxygen labile S toxin is oxygen stable
• When both are active - most hemolysis occurs in the 5% Sheep’s
blood agar
 Streptococcus pyogenes Diseases

• Diseases – 1
– Pharyngitis
– Impetigo (1)
– Erysipelas (2) 2
– Cellulitis (3)
– Necrotizing fascitis (4)
– Puerperal sepsis
– Toxic Shock 3
– Scarlet fever (5)

4
5
 Sequelae of Strep pyogenes
infections
• Rheumatic fever
– Cause: inadequately treated Strep throat
– Rise in the 1990s due to EIA tests being used as the only diagnostic test for
pharyngitis – EIA tests are @ 60-70% sensitive – and led to children not
receiving antimicrobial therapy. All negative EIA specimens on children
must be confirmed with culture
– Similarity between the proteins of the Strep A and muscle tissue cause
immune system confusion which leads to immune system to attack heart,
joint, and bones
– Anti-streptolysin O measures the amount of antibodies against
Streptococcus group A in the serum

• Glomerulonephritis
– 10-14 days following skin infection or pharyngitis
– Renal disease with inflammation of the glomeruli
– ASO titers will be positive
– Usually resolves with therapy
 Streptococcus agalactiae (Group B)
Lancefield agglutination test demonstrates “B” cell wall CHO

Biochemical tests for identification:

Camp test – performed using a Staph aureus strain with Camp
factor streaked perpendicular to group B Strep, incubate 24
hr. / intensifies toxin and produces arrow shaped hemolysis
Arrow-like
Rapid hippurate hydrolysis -
4 hr identification of GBS - Hippurate disk in
Saline inoculated with Group B Strep
Ninhydrin reagent added for color formation
Positive = Purple Staph aureus

pos Strep group B

 Strep agalactiae [GBS]
• Pathogen of the elderly – mostly bacteremia and urinary tract infection
• Pathogen of neonate – in utero or perinatal organism acquisition during
birthing process
– Early onset – within 7 days
– Late 7 – 28 days from birth process.
• Treatment: Penicillin or Cephalosporin (3rd generation)

• Pregnant women carry organism in the cervix and/or rectal area. All
pregnant should be tested at 35 – 37 weeks of pregnancy.
– Enrichment methods for GBS is standard of practice and must be used
• Swab placed into LIM broth – incubate for 18 hours at 35 ˚C then
subculture onto Blood agar. This broth can also be used as an enrichment
method to enrich for molecular testing.
• Carrot enrichment broth turns orange with growth of group B Strep

Carrot Broth
 6.5% Salt

Enterococcus
• Two most common species Neg Pos
– E. faecium
– E. faecalis
Neg Pos
• No well defined virulence factors
• Possesses the Group “D” CHO in the cell wall

• Biochemical tests:
– Bile esculin positive – growth and black precipitin Bile esculin
– Growth in 6.5% salt
PYR
– PYR positive (Group A beta Strep and Staph
lugdunensis also positive)* + -
– E. faecium = arabinose fermentation positive
– E. faecalis = arabinose fermentation negative PYR
 Enterococcus
• Variety of infections / Pathogen of opportunity
• Intestinal normal flora
• Cause UTI, bacteremia, and abdominal infections
• Antimicrobial therapy:
– Natural resistance to cephalosporin antibiotics
– Ampicillin plus Aminoglycoside can be synergistic for therapy in
cases of endocarditis

• Unique susceptibility issues

– Acquired resistance to vancomycin known as vancomycin resistant
enterococcus or VRE
• Van A (E. faecium) resistance genes
• Van B (E. faecalis) resistance genes
 Alpha hemolytic
Streptococcus
Streptococcus pneumoniae
Gram stain = Gram positive bullet (lancet) shaped cocci in pairs
Polysaccharide capsule = virulence factor, antiphagocytic

Identification:
– Bile soluble – colonies dissolve in sodium deoxycholate (bile)
– Optochin sensitive – 14mm or greater zone of inhibition around the
6mm optochin disk – “P” = pneumococcus

Colonies dissolved

Inhibition NO
>=14mm Inhibition
 Streptococcus pneumoniae
• Upper and Lower respiratory tract infection, sepsis and
meningitis
• Multi-serotype vaccine aids preventing invasive infections
• Susceptibility issues:
– Acquired Resistance to Penicillin due to Penicillin binding
proteins (PBP)
– Minimum inhibitory concentration (MIC) testing necessary
to detect resistance to penicillin – in broth or Etest method
– Testing of CSF isolate a result of
– Penicillin = >2 mcg/ml is resistant
• Therapy – MIC
– If susceptible 1st line therapy is either MIC
– Penicillin or 3rd generation Cephalosporin
 Viridans Streptococcus
Several species of alpha hemolytic Streptococcus are NF in
mouth and upper respiratory tract
S. mutans S. salivarius S. sanguis S. mitis
• Bile esculin slant = negative
• Not bile soluble Viridans Streptococcus
• Optochin resistant with zone size <=13 mm
• Cause 30 – 40% cases of sub acute endocarditis / native valve
• Can cause abscess and various infections throughout the body
especially in the immune suppressed host
• Variable susceptibility patterns, some isolates with elevated
MICs to Penicillin
 Viridans Streptococcus
unique species
• Streptococcus anginosis group: includes
– S. anginosus S. constellatus S. intermedius
• Normal flora in human mouth
• More virulent than “normal” viridans
Streptococcus, perhaps due to capsule
• Cause deep tissue abscess, bacteremia,
endocarditis, intra abdominal infections…
• Variable susceptibilities – so best to do
susceptibility testing
 Nutritionally Variant Streptococcus
• Vitamin B6 (pyridoxal) deficient – so will not grow on media without B6
supplementation

• Will grow in a patient blood culture bottle due to vit B6 in patient’s blood
• Will not subculture onto 5% Sheep’s blood agar plate ( no vit B6)
• Requires Staph aureus streak (supplies vitamin B6)
• Nutritionally variant Strep will satellite @ S. aureus streak (see pix)

• There are 2 species:

– Abiotrophia spp
– Granulicatella spp

• Endocarditis –
– More destructive to valve than Satellite streptococcus
than “regular” viridans Strep
– Higher MIC’s to Penicillin
Gram Negative Cocci
Neisseria
Moraxella
 Gram Negative Cocci
• Neisseria species and Moraxella catarrhalis
– Small kidney bean shaped cocci in pairs
– Oxidase enzyme positive
• CTA (Cysteine Trypticase Agar) carbohydrate fermentations
for identification – glucose, maltose, lactose, sucrose
– N. gonorrhoeae Gluc + Mal - Lac - Suc -
– N. meningitidis Gluc + Mal + Lac - Suc -
– N. lactamica Gluc + Mal + Lac+ Suc-
– M. catarrhalis all negative Dna’ase +
• N. gonorrhoae will NOT grow on 5% Sheep’s blood agar
• N. meningitids will grow on 5% Sheep’s blood agar
 Miniature CHO fermentation reactions for Neisseria
and Moraxella catarrhalis

control +

Compare (+) yellow reactions to negative (red) control well

Oxidase enzyme spot test:

Detects presence of enzyme cytochrome
oxidase Add reagent
N,N dimethyl-p-phylenediamine oxalate
to filter paper with organism
positive = purple/ blue

+ +
Full size tube CHO fermentation test
 Neisseria meningitidis
• Meningitis in children and young adults
• Hallmark is petechia (organisms crowd into capillaries) tissue
necrosis and DIC (endotoxin) /infection can be rapidly fatal
• Carriage in Nasopharynx/ Susceptible to Penicillin
• Capsular polysaccharide is the primary virulence factor
• Complement deficiencies in 7,8,and 9 can predispose to
chronic type disease
• Adrenal necrosis is referred to Waterhouse
Friderichsen syndrome
• Type C is the current endemic strain in the USA – 1*problem
in college Freshmen living in dorm
• Immunization available for all serotypes except serotype B
 Neisseria gonorrhoeae
– Acute urethritis, endocervix, ocular, rectal, oropharynx,
septic arthritis
– 10 – 20 % female ascend to PID but only 0.5% disseminate
– Gram stain of urethral discharge useful for male diagnosis,
however, cervix Gram stain not specific for females
– Transport for culture – charcoal swabs, No refrigeration
– Media: Selective Thayer Martin or Martin Lewis agar
– Amplification methods [PCR] increase sensitivity of
detection and superior to culture
– Produces a beta lactamase enzyme and also Chromosomal
resistance : Therapy: Ceftriaxone + Azithromycin or Doxycycline to prevent
resistance
 Neisseria species
gram negative diplococcus – intra and
extra cellular
Primary reason why female Gram stains
Cannot be trusted.

Acinetobacter species –
Cocci are larger and more round
than Neisseria, not usually in pairs –
normal flora in female genital tract
 Moraxella catarrhalis
• Pneumonia, ocular, sinusitis, otitis media
• Gram stain of sputum can be helpful in
diagnosis of Moraxella pneumonia
– Polys with gram negative diplo-cocci
• Hockey puck colony – able to push
around on agar surface
– Oxidase enzyme positive
– DNA’ase enzyme positive
– Resistant to ampicillin by beta lactamase enzyme
production – Cefinase test can be used
– Therapy: Augmentin or 2nd or 3rd generation Cephalosporin
Gram Positive Rods
Corynebacteria
Bacillus
Listeria
Erysipelothrix
 Corynebacterium
•Over 20 species, most not pathogenic
•Gamma hemolytic gray colonies
•Catalase +
•Diphtheroid morphology – Gram positive
Chinese letter forms
•No spores produced
 Corynebacterium diphtheriae
• Agent of Diphtheria
• Diphtheritic adherent pseudo membrane produced in throat
• Phage mediated exotoxin is distributed from the membrane
causing respiratory paralysis (virulence factor)
• Toxin detected by Elek immuno-diffusion test
• Grows well on 5% Sheep’s BAP Elek plate
• Selective medium Cysteine Tellurite agar
– Produces black colonies with brown halos
• Grow on egg containing Loeffler medium then stain
with methylene blue to observe metachromatic
granules - colorful storage granules characteristic of C.
diphtheria
Tellurite agar
Metachromatic granules
 Other Corynebacterium
• Corynebacterium jeikeium –
– normal skin flora bacteria / thrives on lipid
– Infects patients with plastic catheters and indwelling
devices by tunneling into the device from the skin
– Biofilms are formed on the plastic, protecting the
organism from antibiotic therapy
– Very resistant to most antibiotics
– Susceptible to vancomycin and tetracycline only
• Corynebacterium urealyticum – Red is (+) for
Urease reaction
– Rapidly urease positive diphtheroid
– Urinary tract infection in post renal transplants
– Resistant to many antibiotics – vancomycin susceptible
 Bacillus species
• Large gram positive rods boxcar shaped with square
ends
• Can over-decolorize easily and appear reddish
• Spores produced – causes clearing in rod
• Catalase enzyme positive
• Most are motile
 Bacillus anthracis
• Agent of Anthrax
• Natural infection of herbivores – infected animals can have
fatal infection and contaminate the environment for years
• Virulence factors: anthrax toxin and capsular polypeptide
• Skin (Woolsorters’ disease) is the most common
presentation
• Unique black eschar skin lesions
• Pneumonia, sepsis, and meningitis
uncommon
 Bacillus
• Bacillus anthracis culture:
• Medusa head colonies on BAP
– non-hemolytic on 5% Sheep’s blood agar
• Non-motile
• Penicillin susceptible
• With these reactions – Alert public health
department for assistance
• Bacillus cereus – food poisoning with rapid
onset (1 – 6 hr) of vomiting
• Preformed emetic toxin in food
• Fried rice is one of the most common food sources
• Traumatic wound infection from contaminated soil
• Beta hemolytic colony on Sheep’s blood agar
• Motile
Listeria monocytogenes
• Small gram positive rod
• Catalase positive / No spores produced
• Subtle beta hemolysis on Blood agar
• Tumbling motility on wet mount
• Umbrella motility in tube media
• More motile at 25˚C than 35˚C
• Cold loving – Grows well at 4˚C and reason it is
abundant in refrigerated foods
• Found in dairy products and deli case meats
• Infections: Bacteremia in pregnant women and
can induce still births
• Infections: Bacteremia and CNS in immune suppressed
• Ampicillin is drug of choice/ resistant to Cephalosporins
 Erysipelothrix rhusiopathiae
H2S production on
Triple sugar iron agar (TSI)
• Small gram positive rod
• Catalase enzyme negative
• Alpha hemolysis on blood
– Only G+R that produces hydrogen
sulfide (H2S) in a TSI agar slant
– Human skin infection acquired from swine with erysipelas or
poultry
• Infection most common in butchers
– Bacteremia uncommon
• most common in drug addict
• High % of endocarditis with extensive damage to valve
– Intrinsically resistant to Vancomycin
Gram negative bacilli

Enterics
Non fermenters
Fastidious
 Gram negative cell
Wall
Rod shaped
Enterobacteriaceae
Simple flow chart Non fermenters
Enteric Gram Negative Bacilli
Identification Glucose is not fermented
Ferment glucose Strategy Oxidase positive and
Almost all are oxidase negative
negative
Nitrate reduced to nitrates
species

Gram negative Bacilli Gram negative Bacilli

Enteric Gram Enteric Gram Do NOT ferment Do NOT ferment
Negative Rod Negative Rod Glucose Glucose
Ferment Glucose Ferment Glucose Do not ferment Do NOT ferment
Ferment Lactose Do NOT ferment Lactose Lactose
Ex. Escherichia Lactose Positive Oxidase Negative Oxidase
coli Ex. Proteus species Pseudomonas Acinetobacter
Burkholderia Stenotrophomonas
 Enterics that Ferment Glucose
• Escherichia coli
– Major aerobic NF in intestine
– #1 cause of UTI [@80%]
– Bacteremia, neonatal meningitis
Green sheen on
– Abdominal cavity infections EMB agar
– Spot indole reaction = positive
Indole positive =
– Green sheen produced on EMB agar
Robin’s egg blue
• Pathogen of diarrhea
– Traveler’s diarrhea - travel to developing countries
– Enterohemorrhagic E. coli [0157:H7 as well as other serotypes]
• Bloody diarrhea usually acquired from eating undercooked cow
meat from an infected cow
• HUS – hemolytic uremic syndrome [hemolytic anemia, Mucoid colony
thrombocytopenia, and renal failure] particularly in children
• Does not ferment sorbitol – most other E. coli types ferment
Sorbitol
 Enterics that ferment Glucose
• Enterobacter species
• Enterobacter cloacae and E. aerogenes most common
• Environmental organism with low pathogenicity
• Enterobacter (Cronobacter) sakazakii associated with
neonatal meningitis

• Klebsiella species –
• K. pneumoniae most common
• Mucoid colony
• Currant jelly sputum in alcoholics due to blood mixed with
capsular polysaccharide in sputum
Enterics that do NOT ferment lactose!
No fermentation = colorless colony on Mac
• Proteus species
Swarming colonies in layers on agar surface
• Indole positive – Proteus vulgaris
• Indole negative – Proteus mirabilis
• Serratia marcescens
– Produces a red pigment that intensifies at room temp
– Causes infections in
• immune suppressed
• Ventilator associated pneumonia
• Bacteremia
 Gram negative bacillus
MacConkey agar

Lactose fermenter

Lactose non
fermenter

Gram stain enteric

bacilli – plump

Lactose fermentation No lactose

fermentation
 Triple Sugar Iron Agar – Detect fermentation of
glucose, lactose and/or Sucrose and the
production of hydrogen sulfide [H2S]

Fermentation= yellow
Gas = Disruption of the agar No fermentation =
red
H2S

Glu/lac/suc Glucose Glucose No CHO

fermented fermented fermented fermentation
with gas only with H2S Non fermenter
 Extended Spectrum Beta Lactamase
Enzymes – “ESBL”
• ESBL enzymes are produced by numerous species of gram
negative rods
– These enzymes confer resistance to Cephalosporins and
synthetic Penicillins
• Plasmid mediated Tem 1 beta lactamase is the most common
ESBL enzyme produced
– Possible plasmid dissemination makes this an infection
control issue
• ESBL producing GNR are usually treated with:
– Carbapenemases – Meropenem, & Imipenem most
common
 Carbapenamase Resistance (CRE)
• Resistance enzymes conferring resistance to
carbapenamase antibiotics (meropenem and
imipenem)
• Most common in Klebsiella pneumoniae –
where this enzyme is known as “KPC”
• Cross resistance to virtually all classes of
antibiotics – so very difficult to treat
• Treatment: Colistin or antibiotic combinations
 Salmonella species
• Salmonella spp.
– Diarrhea with +/- fever – polys in the stool
– Infection from food - Eggs, meats and contaminated
uncooked vegetables, must ingest large #’s of organisms to
make you ill (1,00,000 bacteria), stomach acid protective
– Does not ferment lactose/ produces Hydrogen sulfide
– Selective agars for growth: SS and Hektoen
– Identification based on biochemical reactions & serologic
typing
– Kaufman White serologic typing scheme
• O Somatic (cell wall) antigen – Salmonella group “B”
• H flagellar antigens – 2 phases [h1 & h2] aids in speciation
Salmonella
• Vi capsular antigen – Found in S. typhi only
Salmonella Shigella Hektoen agar –
Agar (SS agar) Salmonella produces H2S [Hydrogen
Salmonella and Shigella are sulfide] producing black colonies
colorless due to lactose not Shigella – green colonies
being fermented Normal flora – orange colored due to
fermentation of lactose
 Salmonella typhi
• Typhoid fever – fever, sepsis, high fatality rate
• Vi capsular antigen surrounds the D cell wall antigen
• Boil solution of organism for 15 minutes to destroy the
Vi capsular antigen and expose the cell wall D antigen
• Serotyping can then be performed for the D antigen
• Moustache of H2S in the TSI tube
– Carrier state in gallbladder
– Ingest organism, it clears the bowel
then in @ 1 week enters
Blood stream & Bone Marrow
 Shigella
• Diarrhea, +/-vomiting, fluid loss, polys and blood in stool ,
tenesmus, usually treated, Quinolone therapy
• Human to human transmission /control with good hygeine
• Low #’s of organisms to make you ill [10 – 100 bacteria]
• Non motile and No H2S produced (differ from
Salmonella) – Green colonies on Hektoen
• Does not ferment lactose
• 4 species based on somatic antigen
– S. boydii Group C
– S. dysenteriae Group A
– S. flexneri Group B
– S. sonnei Group D
 Yersinia enterocolitica
• Diarrhea
• Major reservoir – swine
• Human usually infected by non pasteurized milk
• Causes septicemia in iron overload syndromes
• Unique infection Mesenteric adenitis – RLQ pain
which mimics appendicitis
• Grows well at 4 °C (like Listeria)
• CIN agar (Cefsulodin-irgasan-novobiocin) is
selective for Y. enterocolitica
• Has been associated banked blood infections related
to transfusion of infected products
 Yersinia pestis - Plague
• Category A agent of bioterrorism
• Obligate flea/ rodent/ flea cycle
• Infected Flea bite - leads to Bubonic form/
painful buboes (lymph node swelling)
• Pneumonic form +/- bacteremia spread
• Fatality >=50%
• Endemic Southwestern USA
• Grows on blood agar
– catalase +, oxidase -
– Bipolar staining “safety pin”
 Glucose Fermenters
Oxidase Positive
• Vibrio cholerae TCBS Agar

– Rice water stool/ mucus flecks in diarrhea

– Natural environment is saltwater
– Virulence due to enterotoxin – receptor on epithelial cell –
activates adenyl cyclase – increases cAMP with
hyper secretion of NaCl and H20 – death from dehydration
and metabolic acidosis
– Halophilic bacteria which means it is a salt loving / 1% salt
actually enhances growth
– Curved appearance to gram negative rod
– Selective media – TCBS = thio citrate bile sucrose agar,
colony turns yellow due to sucrose fermentation
Other Vibrio species
• Vibrio parahaemolyticus
• Colony on TCBS medium is green = sucrose negative
• Diarrhea from ingestion of raw fish and shellfish
• Vibrio vulnificus
• Highly virulent Vibrio species – infection leads to formation of
painful skin lesions on lower extremities with muscle necrosis
• Most prevalent in patients with pre existing liver disease
• 50% fatality rate
• Ingestion of raw oysters and shellfish
from coastal waters
• Usuallly Green colonies TCBS
Classic Gram Negative Rod Gram
Stains
Campylobacte Vibrio species
r

Sea gull wings C shaped

Enteric gram negative rod

Plumb and rectangular

 Do not ferment Glucose
Oxidase negative
• Acinetobacter species – isolated from a hospital environment
and human skin, large gram negative cocco-bacilli
– Ac. baumannii is a major nosocomial pathogen
• Glucose oxidizer
• Acquires antibiotic resistance with pressure
– Ac. lwoffi –
• glucose non oxidizer

• Stenotrophomonas maltophilia
– Rapid maltose oxidizer
– Long Gram negative bacillus
– Naturally resistant to many antibiotics
– Gun metal gray pigment
– Nosocomial pathogen – super-colonizer when on long term Imipenem
therapy due to natural resistance to Imipenem
 Glucose non-fermenter
Oxidase Positive
Pseudomonas aeruginosa
• Fluorescent & blue-green
pigment(pyocyanin)
• Grape-like odor
• Growth at 42˚C
– Ps fluorescens/putida – no growth at 42°C
• Major pathogen in cystic fibrosis
– Mucoid strains [polysaccharide capsule]
– in combination with Burkholderia cepacia
– can cause major lung damage
• Nosocomial pathogen –
associated with water & moisture
• Intrinsically resistant to many antibiotics
Glucose Non Fermenters/oxidase +
• Burkholderia cepacia
– Dry , yellow colony
– Oxidase negative
– Problem organism in cystic fibrosis
colonization and infection with extensive lung damage
• Flavobacterium (Chryseobacterium) meningosepticum
– Associated with fatal septicemia in the neonate
– Low virulence
– Environmental source
– Yellow pigment
 Haemophilus species
Haemophilus influenza
– Variety of infections
– Transmission – close contact secretions
– Polysaccharide “B” capsule = virluence factor
– Requires 2 nutritional factors for growth
• X = hemin
• V= NAD (nicotinamide adenine dinucleotide)
• Demonstrate by satellite phenomenon or X/V strips
– Small pleomorphic GNR / Grows on chocolate agar
– Will not grow on 5% sheep’s blood agar
– Requires high level C0₂ [5 – 8%] for growth
– Vaccine targets the H. influenza type B
– Resistance to+Ampicillin by beta lactamase production [15-
20 %], Cefotaxime becomes the antibiotic of choice
 Disk test for Beta lactamase
Detection
• Add bacteria to filter paper impregnated with Nitrocefin or
Cefinase test (yellow colored/chromogenic cephalosporin)
• Incubate at room temp for @ 1 minute

• Positive result is color change from yellow to red - beta

lactamase enzyme breaks down beta lactam ring to form
hydrolyzed red end product
• Detects resistance to Amp/Pen/Cephalosporin in
Haemophilus species, N. gonorrhoea , Moraxella catarrhalis,
and anaerobic gram negative rods
• Does NOT detect the ESBL enzyme
 More Haemophilus species
• H. parainfluenza –
– requires V (NAD) factor only
– Usually normal flora in the upper respiratory tract
– One of the HACEK organisms of endocarditis
• H. (Aggregatibacter) aphrophilus – (new taxonomy)
– No factor requirements for growth
– Infections: abscesses (liver, lung, brain) & endocarditis
• H. ducreyi –
– Requires X factor
– Cause of Chancroid - venereal disease
– “school of fish” appearance on stains
 Demonstrates need for X and V factor
Satellite phenomena

Small pleomorphic GNR

 The HACEK group
• Oral flora can be pathogens of endocarditis caused
by poor detention or dental procedures
• Requires 2-4 days to grow in patient blood cultures
• Haemophilus species = oxidase neg, catalase neg
• Actinobacillus actinomycetom-comitans = oxidase
neg, catalase pos
• Cardiobacterium hominis = oxidase pos
• Eikinella corrodens = oxidase pos, colony pits BAP
• Kingella kingii = oxidase pos, hemolytic on BAP, also
cause infections in small children (septic joints)
 Bordetella pertussis
Whooping cough – three stages of disease
– (1) Prodromal – flu like disease – most contagious stage
– (2) Catarrhal - cough - with classic whoop in small children
Toxin adheres to bronchial epithelial cells and cough
continues until toxin wears off – can be months
– (3) Paroxysmal - recovery phase
• Gram stain = Tiny gram negative coccobacillus
• Inhabits Nasopharynx – specimen source
• PCR most sensitive and specific for diagnosis
• Charcoal containing media for transport and culture –
Regan Lowe Charcoal agar
• Reservoir for infection – young adults due to waning
immunity. Reason behind booster shots for young adults
 Pasteurella multocida
• Primary cause of Cat and Dog bite wound infections
• Human pneumonia from close animal contact
– Infections can disseminate to blood stream
• Found as normal flora in animal’s mouth
• Small gram negative coccobacilli
• Growth on 5% Sheep’s blood agar
– Non hemolytic grey colony
• Will not grow MacConkey agar (big clue)
• Oxidase Positive
• One of very few Gram negative rods
sensitive to penicillin
 Capnocytophaga
• Fusiform shaped gram negative rods –
very pleomorphic
• Gliding motility, fingerlike projections from colonies
• Oxidase negative, Catalase negative
• C. canimorsus – dog bites with bacteremia – high% of these
infections lead to endocarditis

• Other Capnocytophaga species are normal flora in the human

mouth and
• Can infect mouth ulcers induced by chemotherapy –
• Blood stream can be invaded from infected mouth ulcer
Granuloma in bone
marrow
Brucella species

• Disease – Fever of unknown origin, significant joint pain

• Small gram negative coccobacilli – slow and difficult to grow from blood
cultures – intracellular pathogen of the RES system
– Castaneda biphasic blood culture held for 21 days [old school]
– Current: Automated Blood culture systems with growth within 5 days
• Specimens: blood and bone marrow most profitable
– Granuloma formation in bone marrow
• Serology can be used for genera/species diagnosis
• Brucella species in clinical specimen related to animal
species the patient was exposed:
– B. abortus – ingestion of raw cow milk
– B. melitensis – ingestion raw goat milk, feta cheese
– B. suis – contact with pigs B. canis - contact with dogs
 Campylobacter
• Small curved bacilli –
shaped like sea gull wings
• C. jejuni – agent of diarrhea
– Related to undercooked poultry ingestion
– Requires selective media containing antibiotics -Skirrow’s blood agar
– Thrives at 42˚C - can use to selectively culture Campylobacter
– Requires microaerophilic atmosphere (high CO₂, low O₂)
– Significant % Guillain-Barre syndrome as sequelae of infection
• C fetus – Bacteremia in the immune suppressed host / does
not cause diarrheal disease
• C. jejuni – grows at 37˚C and 42˚, hippurate positive
• C. fetus - grows at 37˚C and 25˚C hippurate negative
 Francisella tularensis
• Reservoir – rabbits, rodents, ticks and flies. Humans infected
by bug bites or directly from exposure to animal blood
• Strongly associated with skinning rabbits with bare hands
• Bacteria can penetrate small breaks in skin:
– cause painful skin lesions –
– enlarged lymph nodes –
– leading to bacteremia (ulceroglandular tularemia)
– Pneumonia
• Great hazard to lab workers
• Fastidious small gram negative cocco-bacillus
• Culture media requires cysteine*
 Helicobacter pylori

• Small curved Gram negative bacilli

• Cause of acute gastritis…..gastric adenocarcinoma
• Human to human transmission/ fecal oral route
• H. Pylori, Rapidly!! And strongly urease positive – used for
detection directly from gastric biopsy tissue
• Difficult to grow in culture Histologic exam of gastric
biopsy
• Stool antigen/diagnosis and test of cure ]
Serum antibody for exposure
• Treatment – Antibiotics and acid
suppression Hematoxylin-eosin stain or
Warthin-Starry Silver stain
 Legionella
BCYE

Legionella pneumophila most common species [6 serotypes]

• Requires cysteine in culture medium for growth
• Buffered Charcoal Yeast Extract agar -colonies form in 3-5
days
• Will not show on Gram stain of specimens
• Use silver impregnation stains in tissue
• Pulmonary disease associated with water,
cooling towers, shower heads, stagnant water
• Urinary antigen test will detect L. pneumophila type I
infection only
• Treatment: Erythromycin (macrolide)
 Bacteria without cell walls
• Mycoplasma and Ureaplasma – have cell membranes only! Media and
transport contain sterols to protect the membrane
• Do not form discreet colonies on agar plates – must read plates under a
microscope to visualize organisms [up to 14 days of incubation]

• M. pneumoniae –
– community acquired pneumonia
Ureaplasma
– Serology and PCR for diagnosis
– Cold agglutinins produced

• Genital mycoplasmas Mycoplasma hominis

– M. hominis – fried egg colony, vaginitis, cervicitis, postpartum sepsis,
neonatal infections , pre rupture of membranes
– Ureaplasma urealyticum – rapid urea hydrolysis in broth, NGU &
upper genital tract infection, spontaneous abortion, neonatal
infections
 Difficult to grow
• Bartonella henselae – Diagnose by PCR and/or serology
– Cat scratch disease – exposure to cat and cat excrement
– Bacillary angiomatosis – vascular skin lesion +/- invasion / HIV
• Bartonella quintana – cause of trench fever/ vector is the
body louse/Diagnose by Serology

• Chlamydia trachomatis Serovars L1,L2,& L3

agent of Lymphogranuloma venereum
– Serology / clinical findings – lymphatics and lymph nodes involved
• C. pneumoniae (TWAR agent)- Pneumonia, Diagnose by PCR
• C. psittaci- psittacosis, pneumonia, exotic parrot exposure/
Serology, PCR and epidemiology
 Molecular Testing for Neisseria gonorrhoeae
and Chlamydia trachomatis
• Amplification of DNA by Polymerase Chain Reaction (PCR)
– Urine, Cervix, and urethral most often tested
– More sensitive than any culture based system
• Sensitivity/Specificity @ 96%/99%
• Females most sensitive specimen = cervix
• Males good sensitivity with urine and urethral
• PCR replaced culture methods as “Gold” standard of
detection
Elementary bodies in
Fluorescent antibody stain
Iodine staining
of inclusions in McCoy
Cell culture
 More difficult to grow
• Ehrlichiosis – Rickettsia bacteria
– Zoonotic intracellular pathogens
– Anaplasma spp, inclusion in the PMN
– Ehrlichia spp inclusion in the Monocytes
– Vector = Ixodes tick
– Fever, leukopenia, thrombocytopenia,
– Elevated serum aminotransferases,
– No rash (differs Rocky Mountain Spotted Fever)
• Found in the south central, southeast , midwest USA
• PCR and serology for diagnosis
 Spirochetes
• Borrelia burgdorferi - Lyme’s disease
– Primarily found in NE part of USA
– Vector = Ixodes tick
– Serology and PCR

• Borrelia recurrentis - Relapsing fever

Vector = human body louse
Blood smear observe spirochete
 Spirochetes
• Treponema pallidum
– Syphilis
– RPR and VDRL for antibody detection
– Molecular methods Darkfield from chancre lesion
• Brachyspira – intestinal spirochete found
on the brush border of the intestine,
?? Role in disease
• Leptospira interrogans – Leptospirosis
– Fever with rash and renal involvement
– Urine from rats and other animals contaminate water supplies

Spirochete – Shepherd’s crook

 Unusual Bacterial diseases

• Granuloma inguinale
– Klebsiella (Calymmatobacterium) granulomatis
– Rare STD – causes ulcerative genital lesions
• Streptobacillus moniliformis
– Rat bite fever or Haverhill fever
– L form - cell wall deficient bacteria
– Inhibited by SPS in blood culture media
– Needs serum supplementation to grow, will not grow on
routine bacteriology media
– Infection obtained from rat bite
 Anaerobes
• Anaerobic infections can occur in virtually
every organ and region of the body
• Polymicrobial – both aerobic and anaerobic species in the
infections
• Endogenous organisms (commensal flora) cause most
anaerobic infections
– Due to trauma, vascular or tissue necrosis cutting off the oxygen
supply to the involved tissue
• Surgery plus antibiotics often necessary
• Anaerobic culture collection – must be oxygen free
– Gel containing swabs
– ESwab,
– Evacuated vials (port o cult)/ oxygen free collection
– Do not refrigerate specimens – absorb oxygen and kill anaerobes
 Anaerobes

• PRAS media – pre reduced anaerobically sterile

– Media packaged in oxygen free environment
• Most common media used
– CDC anaerobic blood agar
– Kanamycin-vancomycin blood agar
– Bile Esculin agar
– Thioglycollate broth
– Chopped meat glucose broth
• Anaerobic gas pack jars – incubate cultures in anaerobic conditions
• Wet pack – add 10 ml water to hydrogen and CO2 generating
envelope/ requires palladium coated catalysts – generate heat
• Dry pack – (Anaeropack) Absorbs O2 and generates CO2
• Anaerobic bacteria can be poorly staining with bizarre & irregularly shape
Anaerobic Gram Negative Rods
 Bacteroides fragilis group
Pleomorphic irregular staining gram negative rod
–Grows in the presence of bile
–Esculin positive – turning media black
–Resistant to Penicillin and Kanamycin
–Common normal flora in GI tract
–Infections foul smelling/gas produced
–B. fragilis* - most common
Growth on bile
–B. ovatus Black pigment
–B. thetaiotamicron – indole positive
–B. uniformis B. vulgatus
• Resistant to Penicillin by beta lactamase enzyme
• Metronidazole is one antibiotic of choice
 Gram Negative Anaerobic Bacillus
• Prevotella and Porphyromonas species
– Will not grow in the presence of bile
– Will not turn black on esculin media
– Brick red fluorescence and black pigment
– Normal commensal flora of the upper respiratory tract
– Respiratory tract infections
 Fusobacterium spp.
Long thin gram negative bacilli –
spindle shaped with pointed ends
• Normal flora in upper respiratory tract
• Associated with mouth and respiratory tract abscess
formation and liver abscess
• Vincent’s angina – necrotizing oral infection caused
by Fusobacterium species and spirochetes
• Lemierre’s syndrome - oropharyngeal infection that
leads to thrombosis in jugular vein, septicemia, high
fatality rate, caused by Fusobacterium necrophorum
Anaerobic Gram Positive Rods
 Clostridium species

• Gram positive bacilli (boxcar shaped ) with spores

– Bacilli may over-decolorize and appear red
• Clostridium perfringens
– Predominate anaerobic G+R in intestine
– Double zone of beta hemolysis on BAP
– Lecithinase produced on egg yolk agar
– Reverse camp test positive

Lecithinase
Reverse Camp Test
 Clostridium
• Clostridium botulinum –
– Adult disease – Preformed heat labile toxin
ingested in mass produced or home-canned foods
– Infant disease - spore ingested from product
produced in nature, neurotoxin produced in gut
• Begins with constipation and difficult sucking bottle
• Associated with Honey, soil, household dust
– Life threatening neuroparalytic disease
• Clostridium tetani
– Tetanus
– Gram stained cells appear like Tennis racket
– Toxin enters human due to penetrating skin injury
– Disease occurs in Non-immunized
– Spastic contractions of voluntary muscles, hyper-reflexia, lock jaw
(trismus)
 Clostridium
• C. septicum –
– Bacteremia or Gas Gangrene in patient with underlying
malignancy
• C. difficile –
– Normal Colonization in 5% adults and 70% healthy infants
– Disease = Antibiotic associated colitis,
pseudomembranous colitis due to toxin production
[Virulence Factor]
• Toxin A – enterotoxin causing fluid accumulation
• Toxin B – potent cell cytotoxin
• Binary toxin (Nap1) strain is unique and produces larger amount of
toxins A and B and is more virulence
– Diagnosis of infection:
• EIA methods [toxin A/B] are not sensitive and are discouraged
• PCR methods [toxin B] are more sensitive and for infection
• Culture – Cycloserine, Cefoxitin, Fructose Agar [CCFA]
 Actinomyces
• Branching gram positive bacilli - do not form spores
• Normal flora in the mouth
• Capable of forming sulfur granules
in tissue – often found on normal tonsil
• Actinomyces israelii – associated with oral, thoracic,
and abdominal infections, IUD infections
B
 Branching Gram positive rods
of Actinomyces – antler like

Molar tooth colony

Sulfur granule
 Propionibacterium acnes
• Pleomorphic gram positive rod
• Catalase positive
• Indole positive
• Normal flora - skin, oral, GU and GI
• Potential contaminate in blood cultures
• Pathogen in cerebral shunt infections
• Firmly established as significant cause of prosthetic joint
infection – particularly shoulder joints
• Cultures should be held up to 7- 14 days
• Therapy - Ampicillin
 Bacterial vaginosis Clue Cell

• Clue cells are diagnostic

• Mixed anaerobic/aerobic bacterial infection - the two most common
organisms in vaginosis are:
– Gardnerella vaginalis (aerobic gram variable rod) and Mobiluncus
(anaerobic curved gram negative rod)
– Human blood agar used to culture Gardnerella, it
is beta hemolytic and distinctive morphology
• Nugent score – gram stain scoring system to diagnose
bacterial vaginosis [BV]
– Healthy = Lactobacillus-like Gram positive rods
– Intermediate = mixed bacterial types
– BV = Gardnerella and Mobiluncus on smear