El Laboratorio

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Lactofenol

-Composición: Ácido fénico cristalizado 1 g. Ácido láctico 1 g. Glicerina liquida 2 g. Agua destilada 1 g. A esta mezcla se le puede añadir algún colorante para teñir las preparaciones si se desea. Por ejemplo con azul de algodón (o azul de anilina) se detectan las características cianófilas de las esporas y otras células del hongo.

Reactivo Melzer

-Composición: Yoduro potásico 0'75 g. Iodo 0'25 g. Hidrato de cloral 10 g. Agua destilada 10 cc. En 10 ml. de agua destilada se disuelven 0'75 g. de yoduro potásico, 0'25 g. de yodo y finalmente 10 g. de hidrato de cloral. Con este reactivo podremos observar las reacciones amiloides y dextrinoides de muchas esporas y células microscópicas.

Sulfovainillina

-Composición: Ácido sulfúrico concentrado 2 cc. Vainillina 0'25 g. Agua destilada 2 cc. Este reactivo tiñe de oscuro los cistidios y laticíferes de algunos Lentinellus.

Tiñe algunas células especificas, colorea de marrón, pardo rojizo a casi negro la trama de la familia Hymenochaetaceae denominándolo reacción "Xantocroide". En el género Hapalopilus la trama se colorea de gamuza o violeta fuerte. Así mismo en el género Tomentella el carpóforo se colorea de verde. También es interesante la reacción el los liocistidios del género Tubulicrinis cuyas paredes se disuelven con este producto. También colorea de amarillo algunas hifas y cistidios "reacción crisofila"

Azul de Cresilo

-Composición: Se disuelve de 0'5 a 1 g. de azul de cresilo en 99'5 ml. de agua. se deja en reposo de 5 a 10 minutos y a continuación se filtra. Es un reactivo con el que ciertas esporas y paredes hifales se vuelven de color rojizo o violeta rojizo, al contacto con este reactivo, denominándose esta reacción meta cromática.

Rojo Congo Amoniacal

-Composición: Amoníaco concentrado 2 cc. una pizca de polvo rojo Congo. Es un excelente tinte para teñir las paredes de las células microscópicas. Muy aconsejable para el estudio del material de herbario. Para estudiar el material fresco es aconsejable mezclar el Rojo Congo con agua destilada en igual proporción, con esto evitaremos la excesiva dilatación de los elementos microscópicos.

Productos Básicos más Utilizados

-Productos: Potasa. Sosa. Amoniaco. Estos productos diluidos al 40 % son ideales en la recuperación de material seco, para la observación macroscópica y microscópica. Tiñe algunas células especificas, colorea de marrón, pardo rojizo a casi negro la trama de la familia Hymenochaetaceae denominándolo reacción "Xantocroide". En el género Hapalopilus la trama se colorea de gamuza o violeta fuerte. Así mismo en el género Tomentella el carpóforo se colorea de verde. También es interesante la reacción el los liocistidios del género Tubulicrinis cuyas paredes se disuelven con este producto. También colorea de amarillo algunas hifas y cistidios "reacción crisofila"

Cloranfenicol

ORIGEN......Es un antibiótico aislado a partir de cultivos de Streptomyces venezuelae de una muestra de tierra llevada de Caracas. Fue descubierto por Burkholder en 1948. CONSTITUCION QUIMICA......Fórmula global C11H12Cl2N2O5. E una p-nitrofenil-di-cloroacetamido propanodiol. NOMBRE QUIMICO.........FORMULA MOLECULAR.....C11H12Cl2N2O5......PESO MOLECULAR.....Cloramfenicol - 323.13 ESTRUCTURA QUIMICA.....PREPARACION .....PROPIEDADES: Es un polvo blanco o blancoamarillento, inodoro, intensamente amargo; es poco soluble en agua, muy soluble en alcohol propilenglicol acetona y acetato de etilo. Es prácticamente estable en soluciones neutras o ligeramente ácidas, pero se destruye rápidamente en solución alcalina. DESCRIPCION.....SOLUBILIDAD.....IDENTIFICACION..... Disulevase 10 mg de cloranfenicol en una mezcla de 1 mg de alcohol diluído y 3 ml de solución reactivo de Cl2Ca al 10 %. Adicionar 50 mg de zn en polvo y calentar en baño maría por 10 minutos. Descartar el líquido transparente en un tubo de ensayo y agréguese 100 mg de acetato de sodio anhidro y II gotas de cloruro de benzoilo. Agítese la mezcla por 1 minuto, agréguese 0.5 ml de solución reactivo de Cl3Fe y HCl diluído hasta producir una solución transparente, se obtendrá un color rojo violado púrpura. ESPECTRO ANTIBACTERIANO.....Tiene amplio espectro siendo efectivo contra muchos gram negativos, poco activo contra gram positivos . Es activo contra rikectsias y virus, inactivo contra M. tuberculosis y levaduras. USOS.....Es la droga de elección en el tratamiento de la tifoidea, paratifoidea, infecciones intestinales producidas por salmonellas.

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-Definición de medio de cultivo: substrato óptimo para el mantenimiento y crecimiento de microorganismos en el laboratorio. El medio variará dependiendo del tipo de microorganismo.

Componentes de los medios de cultivo:
- Agua
- Extracto de levadura: fuente rica en vitaminas B; también contiene nitrógeno orgánico y compuestos de carbono.
- Peptonas: es el producto que resulta de la digestión de materiales proteicos. Constituye una fuente principal de nitrógeno orgánico; contiene también algunas vitaminas, y a veces carbohidratos. Distinguimos dos tipos, dependiendo del tipo de digestión que hayan sufrido:
Hidrólisis química (ácida o alcalina)
Hidrólisis enzimática
- Infusiones y extractos: contienen las sustancias solubles en agua de tejidos animales (carbohidratos, compuestos orgánicos nitrogenados, vitaminas solubles en agua y sales). Mientras que un caldo se obtiene por ebullición de los tejidos, una infusión implica un "colado" del líquido obtenido. Un extracto es un concentrado de un caldo.
Agentes solidificantes: permiten solidificar un medio previamente líquido. Pueden ser de:
- agar-agar (a partir de 10-14 g/l es un medio de cultivo sólido; para valores inferiores, medios semisólidos).
- gel de sílice
- geles poliacrílicos
Práctica 1a. Preparación de medios de cultivo bacteriológicos:
MATERIAL:
- Granatorio
- Botes con los diferentes componentes del medio de cultivo.
- Probetas y vasos de precipitado.
- "Peachímetro" digital
- Agitador magnético (con placa calefactora) e imanes recubiertos de teflón.
- Matraces o tubos de ensayo.
- Algodón graso.
- Placas de Petri estériles.
- Los medios de cultivo a preparar son el caldo nutritivo y el agar nutritivo, cuya composición es la siguiente:
Caldo nutritivo:
Peptona 10 g
Extracto de levadura 5 g
Cloruro sódico 5 g
Agua destilada 1000 ml
pH 7,2

Agar nutritivo:
Caldo nutritivo con 15 g/l de agar.
PASOS A SEGUIR:
- Primero calculamos las cantidades necesarias de cada componente del medio de cultivo en función del volumen de medio a preparar. En mi caso, al tener que preparar agar nutritivo en tubo de ensayo con 5 ml por tubo, las cantidades son:
Peptona 0.5 g
Extracto de levadura 0.25 g
Cloruro sódico 0.25 g
Agar 0.75 g
Agua 50 ml
- En una probeta medimos el volumen de agua destilada necesario (los 50 ml). Añadimos un poco de este agua a una matraz o vaso de precipitado, en función de si se va a preparar agar o caldo nutritivo. En nuestro caso, a una matraz.
- Se pesa cada uno de los componentes (excepto el agar) y vamos añadiendo al recipiente con el agua. Añadimos el resto del agua y agitamos hasta que se disuelva completamente.
- Ajustamos el pH del medio a 7,2. Para ello nos valemos de HCl 1N (baja el valor de pH) o de NaOH 1N (aumenta el valor de pH).
- En este caso (agar nutritivo) se añade el agar al caldo ya preparado y lo fundimos por ebullición (todos los grupos con agar nutritivo los fundimos juntos).
- Luego repartimos los medios:
Caldo nutritivo: en tubos de ensayo (5 ml por tubo).
Agar nutritivo: en tubos de ensayo (5 ml por tubo).
Agar nutritivo: dejarlo en el matraz y tapar con algodón graso.
- Esterilizamos seguidamente los medios en el autoclave durante 15 minutos (ver parte teórica que sigue a este protocolo)
- Una vez estéril, pusimos los tubos de agar nutritivo de forma inclinada para su solidificación. En el caso del agar nutritivo esterilizado en un matraz se repartirá de forma aséptica en placas de Petri estériles, que se dejarán solidificar en posición horizontal.
- Azúcares: actuarán como fuente de energía (cuando su proporción es de 1 g/l) o como substrato de prueba (1-2%)
- Sales minerales: podemos distinguir dos tipos, atendiendo a la función que desempeñan. Estos son: Sales para el mantenimiento de la presión osmótica (NaCl).
Tampones: carbonatos y fosfatos (este ultimo más eficaz).
- Indicadores de pH: son sustancias que cambian de color cuando el medio alcanza algún valor determinado de pH. Los más utilizados son:
- Azul de timol (Rojo 1,2 Azul 2,8 Amarillo)
- Púrpura bromocresol (Amarillo 5,2 Anaranjado 6,8 Púrpura)
- Azul de bromotimol (Amarillo 6,0 Verde 7,6 Azul)
- Rojo fenol (Amarillo 6,8 Rojo claro 8,4 Rojo cereza)
- Rojo neutro (Rojo intenso 6,8 Rojo 8,0 Amarillo)
- Azul de bromofenol, rojo de metilo, rojo de cresol, fenolftaleina, etc...
- Indicadores REDOX: son sustancias que cambian de color cuando se producen este tipo de reacciones. Distinguimos:
Azul de metileno (azul-incoloro)
Cloruro de trifenil (incoloro-rojo

Agar Y Peptonas

-AGAR

La palabra "agar" proviene etimológicamente de la lengua Malaya y viene a representar el alga roja del género Euchema.El agar es una sustancia collodial seca que se extrae de diferentes especies de algas rojas particularmente de la clase Gellidium, Gracilaria, Pterocladia y Anthopeltis. Dependiendo de la calidad y de sus aplicaciones el agar se divide en dos tipos: industrial y bacteriológico.El incremento de uso de agar para aplicaciones industriales como comidas (carnes y vegetales enlatadas, dulces, pasteles, helados, etc) ha sido enorme debido a sus propiedades como agente dispersador, estabilizante, espesante y agente gelificante. Debido a sus numerosas ventajas el agar sustituye a la pectina. Ya que es una gelatina de origen animal porque tiene aproximadamente 8 veces más de poder gelificante que la gelatina animal.

PEPTONAS

El término "peptona" se emplea para definir un producto soluble en agua que se obtiene por hidrólisis particularmente de una proteína o varias proteínas. Este material contiene una mezcla de aminoácidos libres, péptidos y proteosas que se mantienen en la solución después de calentarlas a 100º C.
La presencia de metales alcalinos o fosfatos puede causar la precipitación de la peptona a un pH neutral. Por ésta razón las peptonas producidas a un pH neutro se deben de utilizar para las fórmulas de los medios.
Existe una gran variedad de peptonas dependiendo de los requisitos de crecimiento de los organismos para ciertos aminoácidos y péptidos. En general las proteínas empleadas en la producción de peptonas son de dos tipos, proteínas animales (caseína, gelatina, carne) y proteínas vegetales (soy yo). Las peptonas se obtienen a través de varios tipos de procesos de digestión como ácido, alcalino y enzimático. La hidrólisis rompe todas las proteínas y péptidos y produce sólo aminoácidos libres. Al mismo tiempo destruye algunos aminoácidos importantes como triptofan. Las peptonas pueden ser usadas para las bacterias como fuente de energía permitiendo la producción de proteínas de H2S, indol, aminos, etc. En la preparación de medios de cultivo uno debe emplear el tipo de peptona que proporciona las características adecuadas para la prueba. Por ejemplo en la prueba de indol se debe emplear una peptona rica en triptonal (peptona de caseína). Es importante recordar que aparte de los aminoácidos presentes las peptonas contienen otros constituyentes que pueden estimular el crecimiento de ácidos nucleicos, minerales, vitaminas y ocasionalmente carbohidratos como es el caso de la peptona de soy yo.

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