Preanalytical and susceptibility Testing

Proper media for culture of a urethral drainage from a man include:

A sheep blood agar plate inoculated with 0.001 mL of urine grows 70 colonies of Staphylococcus aureus. How many colony forming units per mL of urine should be reported? 

the steam autoclave method of sterilization:

nitrocefin

The chromogenic cephalosporin test using nitorcefin is the most sensitive and specific test for the detection of beta lactamase.

A Staphylococcus aureus isolate has an MIC of 4 ug/mL to oxicillin.There is uncertainty as to whether this represents an oxacillin (heteroresistant) resistant strain or a hyperproducer of beta-lactamase.

Strain           Oxacillin          Amoxicillin-clavulanic acid 

strain A         susceptible       susceptible

strain B          susceptible       resistant

strain C          resistant            susceptible

strain D           resistant            resistant\

Based on the above results for oxacillin and amoxicillin-clavulanic acid, which is heteroresistant 

cation content of the media

(Ph)

When performing a stool culture, a colony type typical of an enteric pathogen is subcultured on a blood agar plate. The resulting pure culture is screened with several test to obtain the following resilts:

TSI:    acid butt, alkaline slant, nogas, no H2S

phenylaninine deaminase: negative

motility: positive

serology typing: (shigella subgroup B)

 The serological typing is verified with new kit and controls. The best course of action would be to:

inoculated onto selective plating media and incubated in reduced oxygen with added CO₂ at 42*C

(micraerophillic atmosphere and selective media are needed for Campylobacter)

An expectorated sputum is sent to the laboratory for culture from a patient with respiratory distress. The gram stain of the specimen shows many squamous epithelial cells (>/lpf) and rare neutrophlis. The microscope appearance of the organisms present include:

moderate gram positive cocci in chains and diplococci

moderate gram-negative diplococci

moderate palisading gram-positive bacilli

This Gram stain is most indicative of :

A diabetic foot swab from a 82 year old woman with recurrent infections is submitted for culture. The Gram stain reveals:

many neutrophils, no squamous epithelial cells

many gram negative bacilli

many gram positive cocci in chains

The physican request that all pathogens be worked up. In addition to the sheep blood and MacConkey agar plates routinely used for wound cultures, the technologist might also process:

A Gram stain from a swab of a hand wound reveals:

moderate neutrophils

no squamous epithelial cells

moderate gram positive cocci in chains

moderate large gram negative bacilli

Select the appropriate media that will selectively isolate each organism.

tryptophan

The indole test is used for detrmining an organisms ability to produce indole from deamination of tryptophan.

beta-galactosidase

Through the action of the enzyme beta-galactosidase , ONPG cleaves into galactose and o-nitrophenol (a yellow compound)

JEMBEC system

The JEMBEC system is a transport and inoculation medium used for direct plating of specimens for N. gonorrhoeae. Chocolate- based selective medium is inoculated with the specimen. This is placed in an impermeable plastic bag with a bi-carbonate sodium citrate pellet that absorbs moisture to generate a carbon dioxide-rich envoroment.

Chocolate agar base containing vancoumycin,

 colistin, anisomycin, and trimethoprim is also known as:

disk diffusion.

The disk diffusion procedure will not differentiate S. aureus strains with reduced susceptibility to vancoumycin (MICs 4-8) from susceptible strains even when incubated for 24 hours.

zones of larger diameter would result

a delay of more than 15 mins between placing the disks on the inoculated plate and incubation permits excess prediffusion of the antimicrobial agent  from the disk. This would result in a larger than expected zone diameter.

cephalothin

cefotetin and cefoxitin are second generation and ceftriaxone is a third generation.

Streptococcus pyogenes-penicillin

susceptibility testing should be performed when susceptibility of the organism cannot be reliably perdicted and resistance is known or suspected. Susceptibilityntesting of penicillin for treatment of S pyogenes does not need to be performed routinely since resistance has not been documented.

Gentamycin

(Gentamycin is an aminoglycoside)

blood

The sensitivity of blood culture is 50-70%. Stool cultures are positive in < 50% of patients and urine cultures are positive even less frequently. Bone marrow has a sensitivity of up to 90%.

rejected as unacceptible

ananerobic bacteria do not cause pharyngitis

Staphylococcus aureus

Some bacteria such as Enterococcus sp, H. influenza and Neisseria gonorrhoeae continualy produce beta lactamase. S.aureus produces beta-lactamase only after exposure to an inducing agent (such as penicillin) C diptheriae and S. pyogenes do not produce beta lactamase. 

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A sputum specimen is recieved for culture and Gram stain. The Gram stain smear from this specimen seen in the image

The technologist best course of action would be to:

Call the patient care area and request a new specimen.

here are several sputum screening systems for acessesing the quality of respiratory specimens. In general, neutrophils are a positive indicator of quality, and squamous epithelial cells are a negative indicator of quality, suggesting oropharyngeal contamination. This specimen contains an abundance of squamous cells, (>10/low power field), and would be unaccepitable for cultue.

Refer to the following:

                                   64    32    16        8         4          2     Control g/mL

gentamycin     Ο   Ο   Ο    Ο    Ο     Ο     positive

Examine the broth microdilution susceptibility test shown above and determine the MIC for Gentamycin.

Schlicter

the serum bactericidal or Schlicter test can be used to acess the activity of patients serum when they are recieving long term therapy for endocarditis or osteomyelitis. High titers of anti bacterial activity in the serum suggest adequate dosing, a nontolerant isolate or normal elimination of the antibiotic.

Quality control results for disk diffusion susceptibility test yield the following results:

aminoglycoside zones too small and penicillin zones too large. This is most likely due to the:

oxacillin disk diffusion

With the exception of the oxacillin disk screening test, disk diffusion is not recommended for testing S.pneumoniae aginst beta-lactam agents. S.pneumoniae does not produce beta-lactamase, so beta-latamase testing would not be useful

nitrofurantoin

certian antimicrobials, such as nitrofurantoin and norfloxacin, are used only or primairly to treat urinay tract infections. These agents should not be reported for pathogens recovered from other sites of infections.

The susceptibility results below are reported on an Enterococcus faecalis isolated from peritoneal fluid:

Ampicillin:     susceptible

vancoumycin: resistant

clindamycin;  susceptible

levofloxacin:   resistant

linezolid:  susceptible

The physican calls questioning the results. Which of the following should have been done before the report was called:   

A D test is performed on an isolate of Staphylococcus aureus to determine inducible clindamycin resistance:

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[image]A 10 year old child with cystic fibrosis persents with cough and shortness of breath. er sputum Gram stain is seen in this image:

Based on the gram stain what would be the best medium and incubation condition to optimze recovery of the organism seen?

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The image depicts a Gram stain of a knee fluid from a patient who has recently undergone knee replacement surgery:

Susceptibility testing is performed on a Staphylococcus aureus isolate from a blood culture with the following results:

oxacillin:     resistant

cefazolin:    susceptible

Clindamycin:  susceptible

erythromycin: susceptible

trimethoprim/sulfamethoxazole: susceptible

vancoumycin: susceptible

What should the technologist do: 

the cefazolin result should be changed to resistant since the oxacillin result is resistant

Oxicillin resistant staphylococci are resistant to all beta lactam agents, beta lactam/beta lactamase inhibitor commbinatons and carbapenams. results for these antibiotics should be reorted as resistant or should not be reported

Abiotrophia defectica

Nutritionally deficient streptococcus such as Abiotrophia defectica do not grow on sheep blood agar without the addition of cysteine or proximity to S. aureus

Streptococcus pneumoniae

optochin susceptibility is used to differentiat S. pneumoniae, which are susceptible , from other  alpha hemolytic streptococci which are resistant

pneumococcus

(Strep pneumococcus is susceptible, other alpha hemolytic strep are resistant)

bile esculin, PYR, bacitracin, and hippurate

bile esculin, PYR, bacitracin and hippurate are biochemical test used in the presumptive or definitive identification of beta-hemolytic streptococci such as S. pyogenes, S agalactiae and Enterococcus

After 21 hours a blood culture from a newborn grows catalase-negative, gram positive cocci, The bacterial colonies are small, translucient and beta-hemolytic on a blood agar plate. Biochemical test results of a pure culture are:

Bacitracin:  resistant

CAMP reaction   positive

bile esculin:  not hydrolyzed

6.5% NaCl:  no growth

Assuming that all controls react properly and reactions are verified , the next step would be to:

preform a streptococcus group typing.

this describes Strep agalactiae. This can be confirmed by streptococcus antigen typing.

growth in 6.5% NCl broth.

Enterococcus has the ability to grow in the presence of high salt concentrations such as  6.5% NaCl broth

A Gram positive coccus isolated from a blood culture has the following characteristics:

optochin susceptibility:                     negative

bacitracin 0.04 units suscepibility:   negative

bile esculin hydrolysis                      negative

hippurate hydrolysis:                        positive

catalase:                                            negative 

The organism is most likely:

Streptococcus agalactiae

Streptococcus agalactiae is catalase and bile esculin hydrolysis negetive and bacitracin and optochin resistant and hydrolyses hippurate

latex antigen typing

The most likley organism is Streptococcus agalactiae. Polysacchride  antigen typing will confirm the identificaton the identification of S. agalactiae and differentiate it from other beta-hemolytic streptococci

CAMP test

S. agalactiae

A sputum culture from an alcoholic seen in the ER grows gray, mucoid, stringy colonies on sheep blood agar. The isolates grows redily on MacConkey agar and forms mucoid, dark pink colonies. The colonies yield the following test results:

ONPG:         +

indole           -

Glucose        +

oxidase          -

citrate            +

VP                 +

 The organism is most likely

Klebsiella Pneumonaie

--++

An organism was inoculated to a TSI tube and gave the following reactions:

alkaline slant/acid butt  H₂S, gas produced

An isolate from a stool culture gives the following growth characteristics and biochemical reactions:

MacConkey agar: colorless colonies

Hektoen agar:  yellow-orange colonies

TSI; acid slant/ acid butt, no gas, no H₂S

urea: positive

A TSI tube inoculated with an organism gave the following reactions:

alkaline slant/ acid butt no H₂S, no gas produced 

An organism gave the following reactions:

TSI:    acid slant/ acid butt, no H₂S,  gas produced

indole:        positive

motility:      positive 

citrate:    negative

lysine decarboxylase:    positive

Urea:      negative

VP:  negative

This organism most likely is

Escherecia coli

++--

>100,000 CFU/mL of a gram- negative bacilli were isolated on MacConkey from a urine specimen. Biochemical results are as follows:

glucose:                     acid, gas produced

indole:                        negative

urea:                           positive

TDA:                          positive

H₂S:                           positive

The organism is most likely:

A urine culture had the following culture results:

sheep blood             swarming

Columbia CNA :     no growth

MacConkey:         1. >100,000CFU/mL nonlactose-fermenter

                              2.> 100,000 CFU/mL nonlactose-fermenter with red pigment

The isolates from MacConkey agar had the following biochemical reactions.

TEST              isolate1                 isolate2

TSI                alk/acid                 alk/acid

urea               positive                  negative

TDA              positive                 negative

H₂S                positive                 negative

The organism is most likely
     

An 8 year old girl was admitted to the hospital with  a three day history of fever, abdominal pain, diarrhea, and vomiting. A stool culture grew many lactose- negative colonies that yiealded the following test results.

oxidase:       negative

TSI              acid slant/acid butt

indole:         negative

urease:         positive

ornithine decarboxylase:         positive

H₂S:            negative

motility at 25*C                       positive

(citrobacter+)KCN, malonate, beta-galactosidase,

 (salmonella+)lysine decarboxylase

A fecal specimen, inoculated to Xylose lysine deoxycholate (XLD) and Hektoen enteric (HE) produced colonies with black centers. Additional testing results are as follows

 TEST      |  result             serological test       result

glucose     positive           polyvalent           no agglutination

H₂S           positive            Group A             no agglutination

Lysine decarboxylase po  Group B             no agglutination

Urea          negative           Group C             no agglutination

ONPG      negative            Group D             no agglutination

Indole       positive             Group V            no agglutination

The most probable identification is:

A 10 year old boy was admitted to the emergency room with lower right quadrant pain and tenderness. The following laboratory resuts were obtained:

                    Paitent value         normal range

% segs               75%                       16-60%

WBC count        200                       13

The admitting diagnosis was appendicitis. During surgery the appendix appeared normal; an enlarged node was removed and cultured. Small gram negative bacilli were isolated from  the room temperature plate. The organism most likely is:  

A clean catch urine sample from a nursing home patient is cultured using a .001 mL loop. It grows 67 colonies of a lactose fermenter that has the following biochemical reactions:

TSI: acid/acid

Oxidase: negative

Motility: positive

Indole: negative

Citrate: positive

VP: positive

Lysine decarboxylase: negative

Orinthine decarboxylase: positive

Urea: negative

What should the microbiologist report?

A 64 year old man with lymphoma has a positive blood culture at 18 hours incubation. The organism is a nonlactose fermenting gram-negative bacillis on MacConkey agar. Further testing gives the following reactions:

oxidase: negative

TSI: alk/acid (no H2S)

motility : positive

indole: positive

citrate positive (morganella is citrate neg)

orinthine decarboxylase: negative

urea: positive

PAD: positive

VP: negative

The genus is:

The stock cultures that needed for quality control testing of deamination activity are:

(PAD)

A patient with a nosocomial pneumonia has a sputum gram stain that shows many neutrophils and numerous small gram negative cocobacilli. The organism grew in 24 hours as a mucoid hemolytic colony on blood agar and a colorless colony on MacConkey agar. The organism had the following characteristics :

oxidase : negative

catalase: positive

nitrate: positive

ONPG: negative

orinthine decarboxylase: negative

lysine decarboxylase: negative

X factor no growth

V factor no growth

X and V growth

Haemophilus influenza needs X and V both.

chocolate has X and V

A Camplobacter species isolated from a stool culture gives the following biochemical reactions

nalidixic acid:  susceptible

cephalothin: resistant

hippurate hydrolysis: positive

Oxidase: positive

Catalase: positive

A genus that is found in soil and water and causes infection in immunocompromised patients has the following characteristics:

sheep blood agar: violet pigment

MacConkey agar: growth

42*C incubation: growth

Oxidase: Postive

OF glucose: fermenter

indole: negayive

The Genus is:

Chromobacterium

the purple color on blood gives this away

Multiple blood cultures from a patient with endocarditis grew a facultative anaerobic, pleomorphic gram- negative bacilli with the following characteristics:

hemolysis; negative

MacConkey agar:  no growth

Sheep blood agar: growth in 5%-10% CO2

chocolate agar: : growth in 5%-10% CO2

catalase : negative

V factor: not required

X facter: not required

oxidase: negative

Nitrite:   positive

Indole:    negative

The most likely identification is:

A gram negative bacillus with bipolor staining was isolated from a wound infection caused by a bite from a pet cat. The following characteristics reactions were seen:

oxidase: positive

glucose OF: fermentive

catalase: positive

motility: negative

MacConkey agar:    no growth

Which of the following is the most likely organism?

Bordetella bronchiseptica

pasturella multicida does not grow on MacConkey,and  is not motile

Aeromonas hydrophilia

Wounds contaminated with water

An aerobic, gram negative cocobacillus was isolated on Bordet-Gengou agar from a nasopharyngeal swab 48 hours after culture from a 6 month-old infant with suspected pertusis. The organism exhibited the following characteristics:

MacConkey agar: no growth

Urea:  negative at 4 houes, positive at 24 hours

Oxidase negative

catalase: positive

The most probable identification of this isolate is:

Bordetella pertussis

The oxidase and urea reactions differentiate Bordetella pertussis aand B parapertussis.

B pertussis is oxidase positive and urea positive

rapid hydrolysis of urea

bordeletella bronchiseptica is urease positive in 4 hours.

culture was not incubated long enough

Media for isolation of Legionella should be incubated 235-37*c FOR AT LEAST 7 DAYS.

Production of indophenol oxidase.

Both Acinetobacter and Moraxella display resistance to penicillin, and some species grow on MacConkey agar. Acinetobacter are oxidase negative, and Moraxella are oxidase positive

Growth at 42*C, pyocyanin, gelatinase production.

gelatinase production separates Pseudomonas putida (negative) from Pseudomonas Fluorescence (positive)

A child was bitten on the arm by her sibling and the resulting wound grew a slender gram-negative bacilli that has the following characteristics:

growth on SBA:  colonies that pit the agar

colonies odor: like bleach

Catalase:  negative

oxidase: positive

TSI: no growth

The identification of this organismis:

Eikenella corrodens

Pitting the agar and smell of bleach

colonies are large and spreading after 2 days.

Capnocytophagia requires increased CO2 , fermenta glucose, sucrose, and lactose, and is a gram negative bacillus. Capnopcytophagia produces characteristic spreading colonies.

A college student attended a beach party where raw oysters and other shellfish were consumed. The next day, he had symptoms of septicemia. The blood cultures grew gram negative bacilli with the following characteristics:

oxidase: positive

MacConkey agar: pink colonies

O/129 (ug):  susceptible

The most likely organism is:

Vibrio vulnificans

vulnificans is the only vibrio that ferments Lactose

oxidase

Stenotrophomonas is oxidase negative while most other nonfermenters are ocidase positive

A 17 year old female with cystic fibrosis is diagnosed with pneumonia. A sputum sample grew gram negative bacilli with yellow, smooth colonies that have the following biochemical reactions:

oxidase: positive

TSI: alk/alk

glucose: oxidized

fluorescence: negative

Lysine decarboxylase positive

The most likely organism is:

Burkholderia cepatia

Berholderia cepatia is associated with respirtory infections  in cyctic fibrosis patients, and the biochemicals are typical for this organism.

requirement  of 5%-10% CO2 for growth.

The HACEK group of organisms are gram negative bacilli that require increased CO2 for growth. They are commonly associated with endocarditis, and include Haemophilus species (especially H.Aphrophilus), Actinobacillus actinomycetmcomitans, Cardiobacterium hominis, Eikenella corrodens and Kingella species.

The Laboratory recieves a blood culture from a veterinarian who has been ill for many weeks with fevers in the afternoon and evenings, arthritus, and fatigue. The blood culture is positive after 5 days , and the organism has the following characteristics:

Gram stain: small gram negative cocobacilli

sheep blood agar: growth after 48 hours with small, smooth, raised colonies

What should the microbiologist do next:

The following results were obtained from a culture of unknown origin:

Gram stain: gram negative diplococci

indophenol oxidase : positive

glucose: poositive

Maltose:  negative

Sucrose: negative

The most likely source of the specimen would be the:

Moraxella catarrhalis

(all other niesseria ferment carbohydrates)

Neisseria gonorrhea

One of the less commonly used methods for identification of neisseria gonorrhoeae grown in culture is coagglutination

tetramethyl-phenylenediamine

(they are all oxidase positive)

A vaginal smear is submitted for a gram stain for niesseria gonorrhoea. The technologist finds the following results on the gram stain:

many white blood cells

few epithelial cells

many gram postitve cocci

few gram negative diplococci

few gram positive cocci in chains

The technologist should :

lactose degradation

Neisseria lactamica ferments lactose, Neiserria meningidis does not.

Carbohydrate negative

(asaccharolytic)

An aspirated specimen of purulent material was obtained from a brain abcess. After 24 hours incubation , pinpoint colonies grew on sheep blood and small yellowish colonies grew on chocolate. Gram stain of the organism showed gram negative cocci. Results of carbohydrate degradation studies were as follows:

glucose: acid

maltose: acid

sucrose: acid

lactose: negative

Additioonal testing revealed that the organism was oxidase positive and beta-galactosidase negative. The organism is most likely Neisseria:

sicca

the only one listed that ferments glucose, maltose, and sucrose

****************GPB**********************

A gram stain of organisms on Loffler agar showed pleomorphic gram-positive bacilli.The organism should be subcultured to:

potassium tellurite

(corynebacterium diptheria)

An organism recovered from a sputum has the following characteristics:

Culture:  growth at 7 days on lowenstein-jensen agar, incubated under aerobic conditions with CO2  at 35*C.

Gram-stain:  delicate branching gram-positive bacilli

acid-fast staining:   Branching, filimentous , "partially" acid fast bacterium

These results are consistant with which of the following genera?

a positive toxigenicity test In order to determine if an isolate of Corynebacterium diptheria produces toxin,

testing for the presence of diptheria toxin must be performed using methods such as the Elek test or PCR

H₂S production

the only one

esculin positive

Ummm...its a GPB???

Bacillis antracis.

NON-HEMOLYTIC and NON-MOTILE

Norcardia asteriodes.

*****************ANAEROBES*****************

The characteristic that is most commonly associated with the persence of strict anaerobic bacteria and can be taken as presumtive evidence of their presence in a clinical specimen is the:

The following growth results were observed on media inoculated with a foot abcess aspirate and inoculated in 3%-5% CO2.

SBA: 2+ large gray colonies

PEA:  no growth

chocolate:   3+ large gray colonies

MacConkey:  3+ lactose fermenters

trytocase soy broth: gram negative bacilli and gram-positive bacilli

 Biochemicals were set up on the colonies from the MacConkey agar plate. What should the microbiologist do next?

latex agglutionation vs culture on cycloserine cefoxitin egg-fructose agar

(the gold standard for toxin detection)

A 1-2 mm translucent, nonpigmented colony, isolated from an anaerobic culture of a lung abscess after 72 hours, was found to flouresce brick-red inder ultra-violet light. A gram stain of the organism revealed a cocobacillus that had the following characteristics:

growth in bile: inhibited

vancoumycin: resistant

kanamycin: resistant

colistin: susceptible

esculin hydrolysis: negative

indole: negative

The identification of this isolate is:

Bacteriodes ovatus

*****************FUNGI*******************

The major features by which molds are routinely identified are:

Proper amount of reagent is added.

too much india ink blocks transmission of light.

An organism that demonstrates budding yeast cells with wide capsules in an india ink preparation of spinal fluid is probably:

Cryptococcus neoformans

Cryptococcus neoformens is a cause of meningitus in immunocompromised patients, and produces a polysaccharide capsule.

Candida albicans

(Candida albicans produces germ tubes)

Crytococcus neoformans

is a cause of meningitis in immunocompromised patients, and produces brown colonies (due to melanin production) on bird seed agar.

India ink

 Cryptococcus neoformans may not stain well on Gram stain. Rapid identification is important when yeast is dected in CSF the presence of capsules seen in an indian ink preparation would be presumptive for C neoformans.

positive phenol oxidase test.

Cryptococcus neoformans is the only clinically encountered yeast that is phenol oxidase positive.

A urine culture from a patient with a urinary tract infection yields a yeast with the following characteristics:

Failure to produce a germ tube

hyphae not formed on cornmeal agar

urease negative

assimilates trehalose

The most likely identification is:

Candida glabrata

of the yeast listed only Candida glabrata is urease negative and does not produce a germ tube.

A yeast isolate from a CSF specimen produced the following results:

India ink: no encapsulated yeast cells

Cryptococcal antigen: negative

Urea: negative

Germ tube: negative What should the technologist do next?

carbohydrate assimilation

Conventional test such as carbohydrate assimilation , must be performed for definitive identification of the yeast in this  CSF specimen , especially in light of the fact that the yeast is both crypticoccal antigen and germ tube negative.

Cyclohexamide

Cyclohexamide is known to inhibit the growth of some fungal pathogens, including Cryptococcus neoformens

A neonatal blood culture collected through a catheter grows a small yeast, Microscopically, the yeast apear round at one end , with a budlike structure on a broad base at the other end. Growth is enhanced around olive oil saturated discs, The organism isolated is:

Malassezia furfur

Malsssezia furfur cause catheter-related sepsis, requires lipids for growth, and is a small yeast with a wide bud.

chromogenic agar

Chromogenic agar is extremely effective in determining mixed yeast populations in clinical specimens.

repeat the quality control test , but incubate at 35*C

CHROMagar produces the best color development when incubated at 35-37*C

The morphological characteristics of a yeast grown in rabbit plasma are shown in the image :

[image]

The most likely identification of this yeast is:

Candida albicans

Candida albicans produces a tubular structure with no constrictions (germ tubes) when incubated in rabbit plasma

Cryptococcal antigen

Cryptococcal latex antigen test has been proven to be significantlybmore sensitive than staining methods.

culture conversion to yeast form

Several monomorphic molds resemble the filamentous phase of dimorphic molds, so conversion to yeast phase must be performed for identification of dimorphic molds.

Coccidioides immitis

The arthroconidia of Coccidiodies immitis produce alternating arthrospores.

8-14 tuburculate macroconidia

The presence of turberculate macroconidia indicates a presumptive identification of H capsulatum. The identification must be confirmed using nucleic acid probes or exoantigen testing

Sporothrix shencki

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Subculture the mold phase to moist BHI blood media, incubate at 25*C

Dimorphism differentiates Sporothrix from other fungi listed.

Skin scrapings obtained from the edge of a crusty wrist leision  were found to contain thick walled sherical yeast cells (8-15 um in diamater) that had single buds with a wide base of attachment, Microscopic examination of the room temperature isolate from this specimen would probably reveal: 

[image]

round or pear shaped small conidia attached to conidiphores of irregular lenghts.

(blastomyces)

Examination of a fungal culture form a bronchial washing reveals white, cottony areial mycelium. A tease preparation in lactophenol cotton blue shows the structure shown in the image:

[image]

The most rapid test for definitive identification is:

nucleic acid probes

(coccidoides immitis)

transfer a colony to BHI at 35*C

penicillim marneffei

Pennicillim marneffei is a dimorphic fungi that produces a diffusible red pigment.

macroconidia and microconidia

Dermatophites include 3 genera that are generally differentiated by their macroconidia formation.

lactophenol cotton blue

Lactophenol cotton blue is used as the mounting medium, since lactic acid acts as a clearing agent, phenol acts as a killing agent, glycerol prevents drying, and cotton blue gives color to the structures.

Microsporum audounii

The absence of conidia differentiates Microsporum audounii from the other dermatophytes listed.

Microsporum audounii

[image]

Hair that flouresces yellow-green under a Woods lamp indicates the presence of a Microsporum species. The colony morphology and microscopic characteristics  are consistant with M audounii.

zygomycete

Zygomycete grow rapidly and fill the dish with cotton-candy like growth

eschar biopsy

Mucormycosis is an agressive infection where biopsy, curettage or fine needle aspiration is essential for diagnosis.

Pennicillum can best be separated from aspergillus by:

arrangement of conidia and conidiphores

Microscopic morphology (arrangement of the conidia on the conidiophore) is used to differentiate Penicillum from aspergillus.

Paecilomyces

Elongated and tapered phialides (tenpins) are characteristic of Paecilomyces.

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An isolate from a cornea infection had the following culture results :

Sabouraud dextrose: white and cottony at 2 days , rose color at 6 days.

Slide culture: slender cycle shaped microconidia

The most likely  organism is:

Fusarium

Fusarium freuquently cause corneal infections and have sickle-shaped macroconidia

[image]

Pseudalleschereria boydii

Mycetoma,  is a slow-growing bacterial or fungal infection focused in one area of the body, usually the foot. The infection is characterized by an abnormal tissue mass beneath the skin, formation of cavities within the mass, and a fluid discharge. As the infection progresses, it affects the muscles and bones; at this advanced stage, disability may result.

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Fonsecaea pedrosoi

only Fonsecaea pedrosoi produces Clalosporium, Philophora and Fonsecaea types of sporulation simutaneously.

Pseudallescheria boydii

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inhalation

Specimens from fungal culture should be processed in a biological saftey cabinet to minimize the potential for aerosol spread

An improper specimen was submitted for culture.

Early morning sputum specimens are optimal for AFB cultures.

glycerol

(mycobacteria use glycerol as a carbon source in the presence of mineral salts).

cultures are held for an insufficient length of time.

Many species of mycobacterium are slow growing and may take up to 12 weeks for growth.

decontaminate the specimen with sodium hydroxide.

Specimens contaminated by normal flora must be decontaminated prior to innoculation of media; NaOH is the most common decontaminate.

Improper media was used.

Sabouraud dextrose slants are fungal media; mycobacteria media should have been inoculated.

Review the digestion and decontamination procedure.

The concentration of sodium hydroxyide may be too strong to decrese recovery of AFB and lack of contaminated cultures.

The function of

N-acetly-L-cystine and NaOH In the reagent for acid-fast digestion -decontamination procedure is to:

liqufy the mucus

N-acetly-L-cystine (NALC) is a mucolic agent; the concentration of NALC may be increased to digest thick , mucoid specimens. 

quenching agent

The counter stain acts as a quenching agent and reduces the background flourescence of culture debries

Formaldehyde may be produced

(Which is toxic to mycobacteria)

Oxalic acid

The oxalic acid method is superior to alkali methods for processing specimens contaminated with Pseudomonas.

carbol fusion

Mycobacterium form stable complexes with Kinyoun carbol fusion

Tuberculosis

a positive niacin reaction differentiates Mycobacterium tuberculosis from M leprae, M kansasii and M fortuitum.

Tuberculosis

Mycobacterium tuberculosis and Mycobacterium bovis cannot grow in the presence of NAP.

niacin production

 A positive niacin test differentiates Mycobacterium tuberculosis from most other mycobacteria.

tuberculosis

Growth rate, colony morphology,and niacin reduction differentiate Mycobacterium tuberculosis from other mycobacteria 

rough, nonpigmented colony, slow growth at 37*C, niacin production-positive, nitrate production positive

tuberculosis

Growth rate, colony morphology,and niacin reduction differentiate Mycobacterium tuberculosis from other mycobacteria

Capacity to withstand intra-cellular digestion by macrophages.

Mycobacterium can remain viable but dormant in macrophages for many years.

bovis

Mycobacterium can remain viable but dormant in macrophages for many years.

nucleic acid amplification test

(PCR)

Several commercial PCR tests are available that identify Mycobacterium tuberculosis from positive respiratory specimens.

Perform molecular fingerprinting on both slides.if the molecular finger print is the same on both slides, the lab should review its processing procedures.

If the molecular fingerprint is the same, the smear neagtive culture sould be considered a false positive, and the laboratory should review its processing procedures

rifampin

The primary drugs for the treatment of Mycobacterium tuberculosis include ionizid, rifampin, pyrazinamide, and ethambutol.

Contamination by water organisms

Mycobacterium gordonae is widely distributed in soil and water., and is generally considered nonpathogenic.

Mycobacterium scrofulacem

Mycobacterium scrofulacem is a scotochromogen.

scotochromogens

Scotochromogens produce deep yellow or orange pigment when grown either light or darkness.

photochromogens

Photochromogens produce nonpigmented colonies only when grown in darkness, but pigmented colonies after exposure to light.

detection of weakly acid-fast bacilli in infected tissue

Mycobacterium leprae cannot be cultured in vitro. A clinical diagmosis is made, supported by the presence of AFB in a biopsy specimen.

Lowenstein jensen medium

Lowenstein jensen is an egg based medium used to isolate AFB. The other media are not used for the isolation of AFB

Mycobacterium marinum

Mycobacterium marinum causes infections as a result of trauma to the skin and exposure to contaminated fish tanks, or salt water. Its optimal growth temperature is 28-30*C

Mycobacterium Xenopi

Mycobacterium Xenopi grows best at 42-45*C, and is not pigmented.

exposed to light for 1 hour

Pigment production is controled by an oxygen-dependant, light-indicible enzyme in photochromogenic mycobacteria.

An acid-fast bacillis recovered from an induced sputum had the following characteristics:

pigmentation: yellow in the dark, turning a deeper yellow-orange after 2 weeks of light exposure

Nitrate reduction: negative

Tween hydrolysis: positive at 5-10 days

urease: negative

Based on this information, the organism is most likely Mycobacterium:

gordonae

The characteristics given differentiate Mycobacterium gordonae from the other mycobacteria listed. M szulgai and flavescens are nitrate production positive, and M scrofulaceum is negative for Tween hydrolysis.

Tween hydrolysis

Tween hydrolysis differentiates Mycobacterium gordonae (Tween positive)  from M Scrofulaceum (Tween negative)

DNA probe

Mycobacterium avium  and M intracellular have the same biochemical pattern, but can be differentiated by the use of DNA probes.

Mycobacterium fortuitum

Rapidly growing mycobacteria such as Mycobacterium fortuitum may be <10% acid fast and may not stain with florochrome.

haemophilium

 Mycobacterium hemophilium grows best at 30*C , requires hemin of ferric ammonium citrate for growth, and causes subcutaneous lesions presenting as multiple skin nodules. 

incubate the cultures an additional 4 weeks.

Mycobacterium malmoense reqiures up to 12 weeks for growth. 

Mycobacterium tuberculosis

Mycobacterium tuberculosis is the only mycobacterium listed that is nitrate reduction positive.

**************Viruses and other Microorganisms**************

Virus transport medium containing penicillin, gentamycin and amphotericin is used to collect and transport specimens for virus culture because this medium:

Cytopathic effect

intra nuclear and intracytoplasmic inclusions

Incubate the culture tubes for 2-3 weeks longer.

(cytomegalo virus is a slow growing herpesvirus and may take up to 3 weeks to grow in conventional viral culture)

papilomavirus

Human papilimavirus (HPV) infects epithelial tissues throughout the body, including skin, larynx, and anogenital tissue. Persistant infection with oncogenic types of HPV and intergration of HPV DNA into the cellular genome is a pathway leading to HPV induced neoplasia , such as cervical cancer.

herpies simplex

Herpes simplex virus is the most common cause of fatal sporadic encephalitis in the United States 

Burkitt lymphoma

The classic clinical syndrome associated with Epstein-barr virus (EBV) infeection is infectious mononucleosis. However in immunocompromised patients, EBV is associated with posttransplant lymphoproliferative disorders, and maligncies such as Burkitt lymphoma

Treponemia pallidum,

Chlamydia trachomatis,Neisseria gonorrhoeae and herpes somplex virus can all be isolated in culture. Direct culture of Treponema pallidum on artificial media has not been acheived. Darkfield microscopy and serological techniques are used to diagnose T.pallidum infection.

Treponemia pallidim

Darkfield microscopy can be performed to visualize Treponema palliidum in genital or skin lesions. Darkfield examination allows for the visualization of T pallidums typical morphology and motility.

Chlamydia trachomatis

The presence of typical intracytoplasmic inclusions are particularly useful in diagnosing  Clamydia tracomatis inclusion conjunctivitus. Multinucleated giant cells are seen  in cells infected with herpes simplex and varicella zoster virus. Cytomegalovirus produces large cells with intranuclear inclusions.

Chlamydia trachomatis.

Iodine staining can be used to detect the glycogen rich cytoplasmic inclusions in Chlamydia trachomatis infected cells. These inclusions stain brown with iodine.

glycogen

Iodine staining can be used to detect the glycogen rich cytoplasmic inclusions in Chlamydia trachomatis infected cells. These inclusions stain brown with iodine.

interfere with cell wall synthesis

Bacterial L-forms are bacteria that lose their cell wall as a result as a result of therapy with cell wall active antibiotics, such as beta-lactam antibotics. Unlike mycplasma species, which permanently lack a cell wall, the lack of a cell wall in L-forms is a result of enviromental conditions. Once beta-lactam therapy is discontinued, an L form has the capability to grow a cell wall

Borrelia recurentis

Relapsing fever is caused by Borrelia recurentis and is transmitted by the human body louse. Relapsing fever is characterized by the acute onset of high fever lasting 3-7 days, interspersed with periods of no fever, lasting days to weeks.

birds

Chlamydia psittaci, the agent of psittacisis, is transmitted to humans via inhalation of nasal secretions as well as infected fecal and feather dust. 

urethirtis and conjunctivitis

Chlamydia trachomatis is a well known cause of sexually transmitted infections. including urethritis and cervicitis, as well as inclusion conjunctivitis and pneumonia in neonates. It also causes trachoma and lymphogranulomas vernereum.

lack cell walls

Mycoplasma are smaller than other bacteria and lack a cell wall, instead they posess a trilaminar cell membrane. Because they lack a cell wall , they cannot be stained with gram stain. 

Leptospira interrogans

[image]

 Leptospira are spiral shaped organisms with hooked ends. They are ubiquitous in water (eg lakes, ponds) and assiciated with renal infections in animals, Leptospirosis is a zoonosis, and humans are usually infected via direct or indirect contact contact with the urine of infected animals (including rats). Between 5%-10% of patients with leptospirosis have the icteric form and develop jaundice, and may develop acute renal failure.

Fletcher semi-solid

The clinical syndrome described is most likely the icteric form of Leptospira interrogans. The most commonly used medium for culture is Fletcher semisolid agar. Cultures are incubated in the dark for up to 6 weeks at 28-30*C and examined weekly for by darkfield microspy.

medium containing 10% serum plus fatty acids

.

Leptospiria cultures are incubated in the dark for up to 6 weeks in amibent air at 28-30*C, and examined weekly by darkfield microscopy. Fletcher semisolid agar contains peptone, bee extract and rabbit serum, which create an enriched medium to support the growth of leptospires.

in the first 7 -10 days of infection.

Leptospiria organisms can be readily detected by culturing blood during the first week of illness. After this time the organisms disapprar from blood agar and are excreated from the urine for up to 1 month.

epithelial cell from the base of the lesion

Since viruses are intracellular organisms, the collection of cellular material is required to be able to demonstrate virus using stains or florescent techniques. Scraping of the base of a vesicle are required to collect cellular material. 

Chlamydia trachomatis

Up to 1/3 of patients with Nessieria gonorrhoeae infection are also co-infected with Chlamydia trachomatis. Patients with identified infection with one organism are usually treated for both infections.