TP MIKRO TRIA

8/4/2019 TP MIKRO TRIA

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Ratri Ariani K.

Ida Parwati


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Mikrobiologi okuler terus berkembang. Perkembangan ilmu pencegahan dan

eradikasi infeksi mata. Infeksi mata berat jmlnya Infeksi mata berhub dengan obat

imunosupresif, transplantasi organ ,

penggunaan lensa kontak, pasca operasikatarak, implantasi lensa mata.


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Infeksi mata: penyebab kebutaan.

Anamnesis Pemeriksaan fisik Pemeriksaan mikrobiologi

Identifikasi kuman

Penangananinfeksi mata

Rencana terapi

Mencegah komplikasi


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Tinjauan pustaka:Anatomi mata Patogenesis infeksi mata Berbagai infeksi mata Pemeriksaan mikrobiologi


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Berbentuk seperti pir, saraf optikus sbg

tangkai.Volume: 30 cc, 1/5 bag ruangannya Bag terbesar: lemak, otot

Diameter: 24,5 mmArteri utama: A. oftalmika, cabang A. karotis

interna.

Vena: V. oftalmika superior, inferior.


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Faktor risiko infeksi mata:- Usia- Jenis kelamin- Status imun- Kondisi sosial ekonomi


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Lahir: S. epidermidis, S. aureus,Corynebacterium sp., Propionibacteriumacnes.

Bertambah usia bakteri Gram (-)Antibiotik jangka panjang perubahan

mikroorganisme tumbuh jamur &

bakteri resisten


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Air mata:- Tdd protein, elektrolit, produk antimikroba

(lisozim, laktoferin, beta lisin, komplemen,

Ig)- Substansi seluler: Limfosit, PMN- Lensa kontak perubahan flora normal diganti mikroorganisme patogen


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Mikroorganisme Spesies Persentase

Bakteri AerobCoccus Gram Positif

Coccus Gram NegatifBatang Gram PositifBatang Gram Negatif

Staphylococcus epidermidisStaphylococcus aureusMicrococcus sp.Streptococcus pyogenesStreptococcus pneumoniae

Streptococcus viridiansMoraxella catarrhalisCorynebacterium sp.Bacillus sp.Proteus sp.Klebsiella sp.

Eschericia coliPseudomonas aeruginosaMoraxella sp.

30-80%3-25%1-28%0-3%0-3%0-1%

2-5%5-83%0,7-4.2%0,4-1%0-0,5%0-1%

0-2%0-2%


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Mikroorganisme Spesies PersentaseBakteri Anaerob

Jamur

Propionibacterium sp.PeptostreptococcusBacteroides sp.Lactobacillus sp.Clostridium sp.

Candida sp.

Alternaria sp.Cladosporium sp.

Aspergillus sp.Penicillium sp.

Helminthosporium sp.Cephalosporium sp.Geotrichum sp.Rhodotorula sp.Fusarium

0-33%0-2%0-1%0-2%0-2%

0,27-8.9%0,99-17,1%0,99-14,2%0,36-9,3%0,63-4,1%

0,31-7,7%0,31-4,1%0,27-4,1%0,27-2,2%1,6-2,0%


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Jenis infeksiBakteriVirus

Jamur

Blefaritis


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Jenis infeksi Bakteri Virus Jamur

Endoftalmitis S. aureusS. epidermidisS. pneumoniaeStreptococcussp.lainP. aeruginosaBakteri Gram negatiflain

Virus HerpesSimpleksVirus Varicella-ZosterCytomegalovirusMeasles virus

Candida sp.Aspergillus sp.Volutella sp.Acremonium sp.

Kanalikulitis ActinomycesPropionibacteriumpropionicum

Dakriosistitis S. aureusS. pyogenesS. pneumoniaeH. influenzae

Dakrioadenitis S. aureus

S. pneumoniae

Candida albicans

Aspergillus sp.


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Blefaritis- BP: apus tepi kelopak mata (aplikator

kapas / kalsium alginat) dibasahi HBSS /normal saline.

- Inokulasi: agar darah, agar coklat, agarMacConkey, Sabouraud dextrose agar

- BP pus: inokulasi pada BA, CA, MA, dan

(Brain heart insusion broth) BHIB.- Pewarnaan


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Konjungtivitis:- Penyebab tersering pada anak-anak

adalah H. influenzae, S. pneumoniae, S.

aureus.- Epidemi konjungtivitis:S. pneumoniae &

H. influenzae.


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- Kanalikulitis: infeksi pada kanalis lakrimalis(Actinomyces israelii , Propionibacteriumpropionicum)

- Dakriosistitis: infeksi pada sakus lakrimalisdapat disebabkan bakteri & jamur

- Dakrioadenitisbakteri patogenik

(S. aureus, Streptococcus sp)


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Selulitis Orbita:- Definisi: infeksi pada jaringan di sekitar

bola mata, infeksi akut, paling sering

disebabkan bakteri.- Berpotensi serius menjalar ke posteriorkomplikasi pada SSP & sinus paranasal.


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Penyebab selulitis orbita:- Bakteri: S. aureus, S. pyogenes ,

S. pneumoniae. H. influenzae .-Jamur: Mucormycosis.

Teknik pembedahan: implantasi lensameningkatkan kejadian infeksi iatrogenik.(Propionibacterium acnes)


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Hal yang perlu diperhatikan pd pengambilanBP:

Dari lokasi infeksi yang tepat, dihindari dari

kontaminasi. Saat pengambilan harus tepat.Jumlah BP harus cukup Alat-alat dan tempat pengambilan BP harus

steril. Pengambilan BP: sebelum diberi

antimikroba.


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BP kerokan (scraping) untuk biakanorganisme intraseluler diperiksa dalam24-48 jam, (bakteri) dan 3-7 hari (virus).

Harus dibiakan pada media yang sesuai sebelum pengambilan spesimen, kulit disekitar mata harus dibersihkan terlebihdahulu.

bedside culture


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Pus konjungtiva diambil menggunakan

aplikator kapas steril. BP dari kedua mata dibiakan secara

terpisah. Pengambilan BP kerokan untuk deteksi C.

trachomatis menggunakan aplikatorkalsium alginat.

Isolasi tidak segera dilakukanmedia CarryBlair, media transpor Stuart (dikirim ke lab.


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- Menggunakan BP yang sangat sedikit.- BPmengandung mikroorganisme yang

mudah mati dibutuhkan media cair.

- Sudah memakai antibiotik topikaldibutuhkan media pengkaya

- Inokulasi bedside culture.


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Alat-alat :bunsen, spatula Kimura, pisau bedahsteril, kaca obyek, anesetsi topikal, media

cair, media padat, aplikator steril kalsiumalginat (untuk biakan bakteri), aplikatorpoliester atau kapas (untuk biakan virus)


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- Inokulasi pada agar darah, agar coklat,Brucella Blood Agar.

- Pus inokulasi pada agar darah, agarcoklat, agar MacConkey, dan BHIB; & dibuat3 buah apusan.


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1. Spatula Kimura dibakar dahulu.2. Meneteskan anestesi topikal.

3. Digunakan bagian tumpul pisau bedah steril

4. BP diambil dari kelopak mata atas & bawah.

5. Diinokulasi pada agar darah, agar coklat, BBA,

C. trachomatissucrose phosphate broth,

virus Hanks balanced salt solution.

6. Dibuat 3 atau 4 buah apusan (pewarnaanGram, Giemsa, pewarnaan imunofluoresen(Chlamydiae).


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1.Aplikator dibasahi HBSS.2. BP diambil dari lower conjunctival fornix

(dari medial ke lateral).3. Aplikator dimasukkan ke dalam 1 ml HBSS.

4. Inokulasi agar darah dan agar coklat dengan100 L HBSS.

5. Agar darah diinkubasi 1 malam (aerob, 37oC),agar coklat diinkubasi 10%CO2, 37oC.

6. Dilakukan hitung kuman, identifikasi bakteri,pemeriksaan sensitivitas antibiotik.7. Pewarnaan Gram


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1-10 CFU/100 L 10-50 CFU/100 L 50-100 CFU/100L

>100 CFU/100 L

S. pyogenes

S. pneumoniaeS. aureusCitrobacter sp.

Enterobacter sp.E. coliKlebsiella sp.Proteus sp.Morganella sp.S. mercescens

N. gonorrheaeNeisseria sp. lain

Moraxella sp.Acinetobacter sp.Achromobacter sp.Haemophilus sp.P. aeruginosaPseudomonas sp. lain

Streptococcus Grup

B ( hemolitikataunonhemolitik).Streptococcus GrupC ( hemolitik, hemolitik ataunonhemolitik).Streptococcus sp.lain.

Moraxella catarhalis

S. epidermidis

CONSMicrococcus sp.Bacillus sp.

Corynebacterium

sp.


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Media biakan Suhu Kondisi

Agar darahBrucella Blood Agar

Agar coklatSDAAgar MacConkeyBHIB

Thioglycolate brothLowenstein Jensen

35oC35oC

35oC25oC35oC25oC

35o

C35oC

AerobAnaerob

5-10% CO2AerobAerobAerob

AerobAerob


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Cepat, sangat sensitif.Teknik pemeriksaan in vitro untuk replikasi

atau ampifikasi DNA hanya dalam beberapajam. 3 tahap: denaturasi, annealing, dan ekstensi.

(satu siklus). PCR digunakan secara rutin untuk mendeteksi

virus Herpes Simpleks, virus Varicella Zoster ,adenovirus, C. trachomatis, dan M. tuberculosis


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- Media padat: agar darah, Brucella Blood Agar,agar MacConkey, SDA, agar coklat.

- Media cair: Brain Heart Infusion,

BrothThioglycolate broth.

- Jika tidak ditemukan koloni bakteri setelah 48 jam 7 hari (25oC) untuk melihat infeksi jamur.

- Biakan bakteri anaerob s/d 2 minggu. Jikaditemukan koloni bakteri identifikasi kuman,

sensitivitas terhadap antibiotik


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Pewarnaan untuk apusan cairan vitreus danaqueus:

pewarnaan Gram, Giemsa, KOH, pewarnaanGomaris Methanamine Silver, Calcoflour

White.

Sebelum diwarnai, apusan difiksasi dahulu

dengan metanol 95% selama 5 menit.


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TERIMA KASIH


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Micrococcaceae


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Micrococcaceae Kingdom: Bacteria Phylum:Actinobacteria Class:Actinobacteria Subclass:Actinobacteridae

Order:ActinomycetalesSuborder: MicrococcineaeFamily:Micrococcaceae

GeneraAcaricomesArthrobacter

CitricoccusKocuriaMicrococcusNesterenkoniaRenibacteriumRothiaSinomonasZhihengliuella

Streptococcaceae
http://en.wikipedia.org/wiki/Bacteriumhttp://en.wikipedia.org/wiki/Actinobacteriahttp://en.wikipedia.org/wiki/Actinobacteria_%28class%29http://en.wikipedia.org/wiki/Actinobacteridaehttp://en.wikipedia.org/wiki/Actinobacteridaehttp://en.wikipedia.org/wiki/Actinomycetaleshttp://en.wikipedia.org/wiki/Actinomycetaleshttp://en.wikipedia.org/wiki/Micrococcineaehttp://en.wikipedia.org/wiki/Micrococcineaehttp://en.wikipedia.org/wiki/Acaricomeshttp://en.wikipedia.org/wiki/Arthrobacterhttp://en.wikipedia.org/w/index.php?title=Citricoccus&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Kocuria&action=edit&redlink=1http://en.wikipedia.org/wiki/Micrococcushttp://en.wikipedia.org/w/index.php?title=Nesterenkonia&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Renibacterium&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Rothia_%28bacteria%29&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Sinomonas&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Zhihengliuella&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Zhihengliuella&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Sinomonas&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Rothia_%28bacteria%29&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Renibacterium&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Nesterenkonia&action=edit&redlink=1http://en.wikipedia.org/wiki/Micrococcushttp://en.wikipedia.org/w/index.php?title=Kocuria&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Citricoccus&action=edit&redlink=1http://en.wikipedia.org/wiki/Arthrobacterhttp://en.wikipedia.org/wiki/Acaricomeshttp://en.wikipedia.org/wiki/Micrococcineaehttp://en.wikipedia.org/wiki/Actinomycetaleshttp://en.wikipedia.org/wiki/Actinobacteridaehttp://en.wikipedia.org/wiki/Actinobacteria_%28class%29http://en.wikipedia.org/wiki/Actinobacteriahttp://en.wikipedia.org/wiki/Bacteriumhttp://en.wikipedia.org/w/index.php?title=Zhihengliuella&action=edit&redlink=1


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Streptococcaceae

Scientific classification

Kingdom: Bacteria

Division: Firmicutes

Class: Bacilli

Order: Lactobacillales

Family: Streptococcaceae

Genera

LactococcusLactovumStreptococcus
http://en.wikipedia.org/w/index.php?title=Zhihengliuella&action=edit&redlink=1http://en.wikipedia.org/wiki/Biological_classificationhttp://en.wikipedia.org/wiki/Biological_classification


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- Deteksi antigen- BP:

swab endoserviks,urine

- + 30 mnt

The Chlamydia Rapid


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The Chlamydia Rapid

Test (Swab/Urine) is a rapid

chromatographic immunoassay

for the qualitative detection ofChlamydia trachomatis in female

cervical swab, male urethral

swab and male urine specimens

to aid in the diagnosis of

Chlamydia infection.

Female SwabMaleSwab

Male Urine

Sensitivity 88.5% 78.4% 90.9%

Specificity 96.7% 92.9% >99.0%


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Malnutrition

Recurrent infections Immunosuppressin agents

for organ transplantrecipients

Chemotherapy for cancer Genetic predisposition Skin damageAntibiotic treatment

Medical procedures PregnancyAIDS

Pneumocystis carinii

Candida albicans Staphylococcus aureus Streptococcus pyogenes Pseudomonas aeruginosa Polyomavirus JC polyomavirus Acinetobacter baumanni Toxoplasma gondii Cytomegalovirus Aspergillussp

Kaposi's Sarcoma caused byHuman herpesvirus 8 (HHV8)

Cryptosporidium Cryptococcus neoformans Histoplasma capsulatum Clostridium difficile


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Immature dendritic cells (iDC) are activated and matured bycommensal bacteria, for example, lactic acid bacteria (LAB). TheseLAB-activated mature dendritic cells (DC) produce cytokines able toactivate NK cell cytotoxicity and induce their proliferation. ActivatedNK cells can in turn, via the release of relevant cytokines, recruit

(GM-CSF) and activate iDC (TNF- and IFN-). Alternatively,activated NK cells can exert an editing of DC by killing some of theiDC. At the same time, the early release of IFN- by NK cellsinteracting with LAB-activated DC, most likely in secondarylymphoid organs such as the mesenteric lymph nodes, is critical forshaping the following adaptive immune response toward a type 1 T

cell response. Remarkably, some LAB display opposite outcomesand could hamper T cell type 1 polarization.


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The classical mechanistic explanantion is illustratedabove. Delayed type hypersensitivity results when an

antigen presenting cell, typically a tissue dendritic cellwhich has picked up antigen, processed it and displayedappropriate peptide fragments bound to class II MHC iscontacted by an antigen specific TH1 cell patrolling the

tissue. The resulting activation of the T cell produces

cytokines such as chemokines for macrophages, other Tcells and, to a lesser extent, neutrophils as well asTNFbeta and IFNgamma. The consequences are a cellularinfiltrate in which mononuclear cells (T cells and

macrophages) tend to predominate. It is usually maximalin 48-72 hours.


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Drug inactivation or modification: for example, enzymaticdeactivation ofPenicillinGin some penicillin-resistant bacteriathrough the production of-lactamases.

Alteration of target site: for example, alteration ofPBPthe bindingtarget site of penicillinsin MRSAand other penicillin-resistant

bacteria. Alteration of metabolic pathway: for example, some sulfonamide-

resistant bacteria do not require para-aminobenzoic acid (PABA), animportant precursor for the synthesis offolic acid and nucleic acidsin bacteria inhibited by sulfonamides. Instead, like mammalian cells,

they turn to utilizing preformed folic acid. Reduced drug accumulation: by decreasing drug permeability

and/or increasing active efflux (pumping out) of the drugs acrossthe cell surface.
http://en.wikipedia.org/wiki/Penicillinhttp://en.wikipedia.org/wiki/Penicillinhttp://en.wikipedia.org/wiki/Beta-lactamaseshttp://en.wikipedia.org/wiki/Penicillin_binding_proteinhttp://en.wikipedia.org/wiki/MRSAhttp://en.wikipedia.org/wiki/Sulfa_drugshttp://en.wikipedia.org/wiki/Para-aminobenzoic_acidhttp://en.wikipedia.org/wiki/Folic_acidhttp://en.wikipedia.org/wiki/Nucleic_acidshttp://en.wikipedia.org/wiki/Semipermeable_membranehttp://en.wikipedia.org/wiki/Effluxhttp://en.wikipedia.org/wiki/Effluxhttp://en.wikipedia.org/wiki/Semipermeable_membranehttp://en.wikipedia.org/wiki/Nucleic_acidshttp://en.wikipedia.org/wiki/Folic_acidhttp://en.wikipedia.org/wiki/Para-aminobenzoic_acidhttp://en.wikipedia.org/wiki/Sulfa_drugshttp://en.wikipedia.org/wiki/MRSAhttp://en.wikipedia.org/wiki/Penicillin_binding_proteinhttp://en.wikipedia.org/wiki/Beta-lactamaseshttp://en.wikipedia.org/wiki/Penicillinhttp://en.wikipedia.org/wiki/Penicillin


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The four most important antibiotic resistance mechanisms are

alteration of the target site of the antibiotic enzyme inactivation


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alteration of the target site of the antibiotic, enzyme inactivation

of the antibiotic, active transport of the antibiotic out of the

bacterial cell, and decreased permeability of the bacterial cell

wall to the antibiotic


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Baird-Parker agar is a typeof agar used for the selective
http://en.wikipedia.org/wiki/Agarhttp://en.wikipedia.org/wiki/Agar


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ofagar used for the selectiveisolation ofgram-positiveStaphylococcispecies.Itcontains lithiumchloride andtellurite to inhibit the growthof alternative microbial flora,while the included pyruvate

and glycine promote thegrowth ofStaphylococci.Staphylococcuscoloniesshow up black in colour with

clear zones produced aroundthem.
http://en.wikipedia.org/wiki/Agarhttp://en.wikipedia.org/wiki/Gram-positivehttp://en.wikipedia.org/wiki/Staphylococcihttp://en.wikipedia.org/wiki/Lithiumhttp://en.wikipedia.org/wiki/Chloridehttp://en.wikipedia.org/wiki/Telluritehttp://en.wikipedia.org/wiki/Pyruvatehttp://en.wikipedia.org/wiki/Glycinehttp://en.wikipedia.org/wiki/Glycinehttp://en.wikipedia.org/wiki/Pyruvatehttp://en.wikipedia.org/wiki/Telluritehttp://en.wikipedia.org/wiki/Chloridehttp://en.wikipedia.org/wiki/Lithiumhttp://en.wikipedia.org/wiki/Staphylococcihttp://en.wikipedia.org/wiki/Gram-positivehttp://en.wikipedia.org/wiki/Agar


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- commonly used for specimencollection in pediatrics, public healthmicrobiology, ophthalmology, foodpathology

- Calcium alginate swabs contain nofatty acids that may be inhibitory to

many strains of gonorrhea.


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A type of blood agar plate in which the bloodcells have been lysed by heating the cells to56 C. Chocolate agar is used for growingfastidious (fussy) respiratory bacteria, suchas Haemophilus influenzae.
http://en.wikipedia.org/wiki/Lysishttp://en.wikipedia.org/wiki/Haemophilus_influenzaehttp://en.wikipedia.org/wiki/Haemophilus_influenzaehttp://en.wikipedia.org/wiki/Haemophilus_influenzaehttp://en.wikipedia.org/wiki/Lysis


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Brucella Blood Agar (BRU) is an enrichednon-selective agar medium. BrucellaBlood Agar is intended for the isolation,quantitation and partial identification

of obligate anaerobic bacteria fromclinical specimens.


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Composition: Peptone - 17 g Proteose peptone - 3 g Lactose - 10 g

Bile salts - 1.5 g Sodium chloride - 5 g Neutral red - 0.03 g

Agar - 13.5 g Water - add to make 1 litre; adjust pH to 7.1

+/- 0.2

MacConkey agar (MAC)


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-The addition of bile salts and crystal violet to

the agar inhibits the growth of most Grampositive bacteria, making MacConkey agarselective. Lactose and neutral red are addedto differentiate the lactose fermenters, whichform pink colonies, from lactosenonfermenters that form clear colonies
http://en.wikipedia.org/wiki/Crystal_violethttp://en.wikipedia.org/wiki/Neutral_redhttp://en.wikipedia.org/wiki/Neutral_redhttp://en.wikipedia.org/wiki/Crystal_violet


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Sabouraud agar is used to culture fungi andhas a low pH that inhibits the growth of mostbacteria; it also contains the antibioticgentamicin to specifically inhibit the growth

ofGram-negative bacteria.
http://en.wikipedia.org/wiki/Fungihttp://en.wikipedia.org/wiki/PHhttp://en.wikipedia.org/wiki/Gentamicinhttp://en.wikipedia.org/wiki/Gram-negativehttp://en.wikipedia.org/wiki/Gram-negativehttp://en.wikipedia.org/wiki/Gentamicinhttp://en.wikipedia.org/wiki/PHhttp://en.wikipedia.org/wiki/Fungi


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Fungal media

S b d
http://en.wikipedia.org/wiki/Raymond_Sabouraudhttp://en.wikipedia.org/wiki/Raymond_Sabouraud


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Sabouraud agar Sabouraud agar is used to culture fungi and has a low pH

that inhibits the growth of most bacteria; it also containsthe antibiotic gentamicin to specifically inhibit the growth ofGram-negative bacteria.

Hay infusion agar Specific for the culturing ofslime moulds (which are not

fungi).

Potato dextrose agar PDA is used to culture certain types offungi.

Malt extract agar Malt extract agar has a high content of peptone and is

acidic. It is essentially used in the isolation of fungalmicroorganisms.
http://en.wikipedia.org/wiki/Raymond_Sabouraudhttp://en.wikipedia.org/wiki/Fungihttp://en.wikipedia.org/wiki/PHhttp://en.wikipedia.org/wiki/Gentamicinhttp://en.wikipedia.org/wiki/Gram-negativehttp://en.wikipedia.org/w/index.php?title=Hay_infusion_agar&action=edit&redlink=1http://en.wikipedia.org/wiki/Slime_mouldhttp://en.wikipedia.org/wiki/Potato_dextrose_agarhttp://en.wikipedia.org/wiki/Potato_dextrose_brothhttp://en.wikipedia.org/wiki/Fungihttp://en.wikipedia.org/w/index.php?title=Malt_extract_agar&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Malt_extract_agar&action=edit&redlink=1http://en.wikipedia.org/wiki/Fungihttp://en.wikipedia.org/wiki/Potato_dextrose_brothhttp://en.wikipedia.org/wiki/Potato_dextrose_agarhttp://en.wikipedia.org/wiki/Slime_mouldhttp://en.wikipedia.org/w/index.php?title=Hay_infusion_agar&action=edit&redlink=1http://en.wikipedia.org/wiki/Gram-negativehttp://en.wikipedia.org/wiki/Gentamicinhttp://en.wikipedia.org/wiki/PHhttp://en.wikipedia.org/wiki/Fungihttp://en.wikipedia.org/wiki/Raymond_Sabouraud


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CFU/ mL = CFU/plate x dilution factor On the plate shown, milk was diluted 1 to

100 (10 2), 1.0 mL of the dilution was platedand 40 colonies formed. Therefore the

count per mL in the milk was: 40 colonies x 102 x 1/1 = 4 x 103/mL


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The glycogen, mucin, and fungi will be stained

purple and the nuclei will be stained blue.

Fixation: 10% formalin.

Solutions and Reagents:

0.5% Periodic Acid Solution:

Periodic acid ---------------------- 0.5 g

Distilled water -------------------- 100 ml


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Schiff Reagent:Test for Schiff reagent: Pour 10 ml of 37%

formalin into a watch glass. To this add a fewdrops of the Schiff reagent to be tested. A

good Schiff reagent will rapidly turn a red-purple color. A deteriorating schiff reagentwill give a delayed reaction and the colorproduced will be a deep blue-purple.

Mayers Hematoxylin Solution:


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Procedure:

1. Deparaffinize and hydrate to water.2. Oxidize in 0.5% periodic acid solution for 5 minutes.

3. Rinse in distilled water.

4. Place in Schiff reagent for 15 minutes (Sections become

light pink color during this step).

5. Wash in lukewarm tap water for 5 minutes (Immediately

sections turn dark pink color).

6. Counterstain in Mayer's hematoxylin for 1 minute.7. Wash in tap water for 5 minutes.

8. Dehydrate and coverslip using a synthetic mountingmedium.


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Results:Glycogen, mucin and some basement

membranes --- red/purple

Fungi ------------------------ red/purple

Background ------------------- blue


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Silver staining is a common special stainingtechnique used in medical laboratories.Gomori's Methenamine Silver (GMS) stain isused for fungi and bacteria. The fungi and

bacteria are turned black, while everythingelse is stained green with Light green SFsolution.


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Shell vial cultures Following inoculation of the specimen, the


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Following inoculation of the specimen, theshell vial was centrifuged at 700 g at room

temperature followed by incubation at 36Cfor 1 h for adsorption. The inoculum wasdiscarded and 1 ml of maintenance medium(MEM with 1 % foetal bovine serum) was

added. The vial was incubated for 24 hrs at36C in a CO2 incubator. The coverslip was

removed, fixed in cold acetone for 30minutes at -70C and stained by an indirect

immunofluorescence assay (IFA) using apolyclonal antibody to HSV-1


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Tube cultures Following inoculation of the specimen into a tube


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culture, the culture was incubated for 1 h at 36Cfor adsorption, the inoculum was removed and 1.5ml of maintenance medium (MEM with 1% fetalbovine serum) was added. Cultures wereincubated at 36C in a CO2 incubator for five days

and observed for the presence of cytopathic effect

(CPE) everyday. Cultures were terminated on thefifth day or as soon as CPE was observed (Fig. 2),whichever was earlier. Cells were scraped from thetube, washed in PBS, pH 7.2 and spotted onto a

sterile glass slide. Smears were air dried, fixed incold acetone for 30 minutes at -70C and stainedby an indirect immunofluorescence assay (IFA)using a polyclonal antibody to HSV-1
http://www.biomedcentral.com/1472-6890/2/1/figure/F2http://www.biomedcentral.com/1472-6890/2/1/figure/F2


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TP MIKRO TRIA

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