Microbiological Testing is a mandatory requirement to ensure the quality of the packaging material which has a direct impact on the quality of a drug. The goal of the packaging material is to protect the contents against external factors such as humidity, light, oxygen or temperature variations. Microbiological Testing of the packaging material is done to ensure that the product protected against external impacts or not. The packaging material itself should neither interact with the contents of the packaging nor should it have a negative influence on the contents. Ideally, there will also be no transfer of ingredients from the packaging material to the drug. I will elaborate on how to conduct microbiological testing on empty bottles, droppers, and tubes. Procedure The procedure to conduct microbiological testing of the packaging material is as follows. A For Total Microbial Count A.1 Method Applied: Pour Plate A.1.1 Fill each container with peptone and pour the contents in a pre-sterilized flask. A.1.2 Aseptically takes 1ml of the sample from (A.1.1) in each of four Petri plates. A.1.3 Add (15 — 20ml) of liquid trypticase soy agar on the sample’s taken in 2 Petri plates for bacterial growth. A.1.4 Swirl the plate gently, cover, after solidification, invert to remove moisture from the plate and incubate at 30° — 35°C for 3 to 5 days. A.1.5 Similarly repeat the step (A.1.3 — A.1.4) for sabouraud dextrose agar (SDA) in other two plates for fungal growth and incubate at 20° — 25°C for 5 to 7days. Evaluation After completion of incubation, count the colony-forming units taking an average of two Petri plates for each agar medium and record the results. B For Pathogenic Bacteria B.1 Staphylococcus aureus and Pseudomonas aeruginosa B.1.1 Aseptically transfer 10 ml of the sample from (A.1.1) into 90ml trypticase soy broth, disperse and incubate at 30° — 35° for 24 to 48 hours. B.1.2 If growth is present in (B.1.1) mix gently and streak on:- i- Mannitol Salt Phenol Red Agar Medium —— Staphylococcus aureus ii- Cetrimide Agar Medium —– Pseudomonas aeruginosa B.1.3 Cover, invert and incubate the dishes at 30° — 35°C for 24 to 48 hours. Evaluation Examine any resulting growth. Staphylococcus aureus Staphylococcus aureus gives yellow colonies with yellow zones on Mannitol Salt Phenol Red Agar Medium. Then perform a gram staining test. Gram Staining Test: It should be positive cocci in the cluster Confirmation To confirm the Staphylococcus aureus Biochemical differentiation test (API Staph) is performed. Pseudomonas aeruginosa Pseudomonas aeruginosa generally gives green colonies on Cetrimide Agar Medium. If fluorescence is checked in ultraviolet light, it will be greenish. Then perform a gram staining test. Gram Staining test: It should be gram-negative rods. Confirmation To confirm Pseudomonas aeruginosa, Oxidase test is performed. Oxidase test Transfer the colony to be tested to an Oxidase Detection Strip using a platinum wire loop. Spread the culture on the strip and observe for up to 5 seconds. A deep blue / violet color indicates a positive reaction. The presence must be confirmed by API 20 NE (Biochemical differentiation test). B.2 Salmonella Species B.2.1 Aseptically add 10ml of the sample from (A.1.1) in 90ml lactose broth, disperse and incubate at 30° — 35°C for 24 to 48 hours. B.2.2 If growth is present mix gently and pipette 1ml portion into vessels containing 10ml of Selenite Enrichment Broth (double strength) B.2.3 Subculture onto any of two plates of each medium by streaking on. Brilliant Green Agar Medium Xylose Lysine Desoxycholate Agar Medium Bismuth Sulfite Agar Medium Cover, invert and incubate the Petri plates at 30° — 35°C for 24 to 48 hours. Evaluation 1.0 On Brilliant Green Agar Medium Salmonella give small, transparent, colorless or pink to white opaque colonies (frequently surrounded by pink to red zone). 2.0 On XLD Agar Medium, Salmonella gives red colonies that are with or without black centers. 3.0 On Bismuth Sulfite Agar Medium, Salmonella give black or green colonies. Confirmation To confirm Salmonella, transfer the suspected colonies with the help of an inoculating wire to a butt slant of Triple Sugar Iron Agar Medium. A first streak on the surface of the slant. Stabb the wire well beneath the surface of the slant. Incubate at 30° — 35°C for 24 to 48 hours. If the slants become alkaline (red) and butt become acidic (yellow), with or without concomitant blackening of the butt from hydrogen sulfide production, it indicates the presence of genus Salmonella. Confirm the results by using API 20E (biochemical differentiation test). B.3 Escherichia coli Transfer quantity of the contents A.1.1 corresponding to 1gm or 1ml to 100ml of enrichment medium (Enterobacter enrich broth Mossel) and Incubate the (EBM) and remaining lactose broth at 35 — 37°C for 24 to 48 hours. If the growth is present in EBM mix gently subculture on two plates of Levine EMB Agar and incubate at 35 – 37°C for 24 to 48 hours. Evaluation On the Levine EMB Agar, colonies suspected of being Escherichia coli appear greenish metallic sheen in reflected light, dark or even black center in transmitted light. Perform the gram staining it should be gram-negative rods. Confirmation The biochemical differentiation test is performed to confirm Escherichia coli (using the API 20 E system). Requirements A. Total Microbial Count < 50 CFU B. Pathogenic Bacteria Acceptance Criteria Staphylococcus aureus —- No growth Pseudomonas aeruginosa—- No growth Salmonella species—- No growth Escherichia coli—– No growth
Microbiological Testing of Drinking Water
Drinking Water Drinking water safety is a major concern throughout the world. We all are well aware that 90 percent of diseases are water born. Therefore drinking water should be free from pathogens. In this article, I will explain how to test drinking water in a microbiology laboratory. Preparation of Apparatus Thoroughly wash and finally rinsed glass apparatus e.g., Petri dishes pipettes, flasks, graduated cylinders with purified water and sterilized in a dry heat oven at 160ºC for 120 minutes or 170º to 180°C for not less than 60 mins. Filter units are thoroughly washed with purified water and wrapped loosely with Parchment Paper. Filter holders, scissors, forceps, with Parchment Paper, and along with wrapped filter units sterilize by autoclaving at 121°C for 30 minutes. Excessive or prolonged heating will damage the filters. It may be convenient to sterilize all the above equipment and apparatus in a suitable metal container. Procedure Total Microbial Count (Pour Plate Method) Using a sterile pipette, aseptically add 1ml of the sample into two Petri plates (run in duplicate). Aseptically add 20 – 25ml of melted R2A Agar (at about 45°C) in Petri dishes containing water samples. Swirl the plate gently, cover after solidification, invert and incubate at 30 – 35°C for 48 – 72 hrs. NOTE: The test should be conducted under LFC. Evaluation After completion of incubation, count the colony-forming units taking an average of two Petri plates for each agar medium and record the results. Membrane Filtration Method (Alternate Method) The sterilized filter of porosity (0.45mm) is aseptically placed on the filter base of the filtration assembly while the ration unit is attached to a vacuum pump. The membrane is first rinsed with 100ml of sterile Drinking water and then pour 1ml of sample to be tested (diluted sample can be tested if the count is expected). Rinsed the filter membrane again with drinking water after filtration of the sample (in membrane filtration technique for drinking water testing quantity can be increased that is instead of 1ml, 50ml or 100ml can be used, if not very high count is expected). Disassemble the filtration assembly, remove filter aseptically and placed to R2A Agar plate, incubate at 30 – 35°C for 48 – 72 hours. Evaluation After completion of incubation, count the colony forming units (the result evaluation of membrane filtration will be the total count obtained from the single filter membrane). Confirmation If the number of viable micro-organisms increased the alert limit than First Streak the morphologically identical colonies which are more in count on Tryptic Soy Agar and incubate at 30 to 35°C for 24 hours. Perform the gram staining. If morphological identical colonies are gram-negative rods confirm them with biochemical differentiation test (API 20 E & API 20 NE). If the colonies are gram-positive cocci confirm them with API Staph. Pathogenic Bacteria Identification Psudomonas aeruginosa Add 50ml of water to 25ml Malachite Green Broth (Triple Strength). Thus the final concentration of inoculated broth will always be single strength. Incubation at 35°C ± 1°C for 24 – 48 hours. From the incubated Malachite Green Broth, subculture onto the one plate of cetrimide agar. Cover, invert and incubate the dishes at 30 – 35°C for 24 – 48 hours. Examine the resulting growth as following Pseudomonas aeruginosa generally gives green colonies on Cetrimide Agar Medium. If fluorescence is checked in ultraviolet light, it will be greenish. Perform gram staining, it should be gram-negative rods. Confirmation To confirm Pseudomonas aeruginosa Oxidase test is performed. Transfer the colony to be tested to an Oxidase Detection Strip using a platinum wire loop. Spread the culture on the strip and observe for up to 5 seconds. A deep blue / violet color indicates a positive reaction. The presence must be confirmed by API 20 NE (Biochemical differentiation test). Salmonella Species Aseptically add 10ml of the sample in 90ml lactose broth, disperse and incubate at 35 – 37°C for 24 – 48 hours. If growth is present mix gently and pipette 5ml sample with double strength portions into tubes containing 10ml selenite cystine broth in the ratio 1:1, mix and incubate at 35 – 37°C up to 24 hours. Subculture on any of two plates of each medium by streaking. a- Brilliant Green Agar Medium. b- Xylose Lysine Desoxycholate Agar Medium c- Bismuth Sulfite Agar Medium Cover, invert and incubate the Petri plates at 30 – 35°C for 24 – 48 hours. Evaluation On Brilliant Green Agar Medium, Salmonella gives small, transparent, colorless or pink to white opaque colonies (frequently surrounded by pink to red zone). XLD Agar Medium, Salmonella gives red colonies that are with or without black centers. Bismuth Sulfite Agar Medium, Salmonella gives black or green colonies. Perform gram staining; it should be gram-negative rods. Confirmation To confirm Salmonella, transfer the suspected colonies with the help of inoculating wire to a butt slant of Triple Sugar Iron Agar Medium. A first streak on the surface of the slant and then stabbing the wire well beneath the surface; incubate at 30 – 35°C for 24 – 48 hours. If the slants become alkaline (red) and butt become acidic (Yellow), with or without concomitant blackening of the butt from hydrogen sulfide production, indicating the presence of genus Salmonella. Coli Form and E. coli Add 50ml of water sample into a sterile, transparent 100ml tube/flask with a screw cap. Attention: Glass apparatus is not shelf fluorescence. Take one snap pack of media Readycult® Coliforms. shortly tap to ensure the granules are at the bottom. Bend the upper part of the snap pack until at break opens. Attention: Do not touch the opening to avoid contamination risk Add the content to the water sample seal the vessel and shake to dissolve the granules completely (broth is clear & yellowish). Incubate 18 – 24 hours at 35°– 37°C. Interpretation of Results Negative——- No Color Change Total Coli Form—–Color Change to Blue-green (No discoloration with shaking) E. coli——Check blue-green colored vessel for fluorescence by the UV lamp. To