MicroBiology Project
by Lakshmi,Introduction
All species of fish, when properly chilled, will stay fresh for longer periods than those that are not preserved in any way. The use of chilling techniques such as ice, therefore, effectively prolongs the length of time available for fishing trips and makes it possible to increase the catch with economic benefits for the vessel and crew. Products brought to market in a well-preserved condition will generally command higher prices, both at wholesale and retail levels, and thus give better returns to the fishing operation.
Given the above, it might be assumed that all types and sizes of fishing vessels would benefit from the use of ice for catch preservation. However, in practice there are limitations. On the smallest types of vessels, such as small rafts and the smallest dugout canoes, there is no space to keep ice until it is needed. However, this may not be a problem as the fishery undertaken by these very small craft usually only lasts a few hours and fish is consumed or sold on a daily basis. In some of these very small fishing craft, owners are aware of the problems of catch deterioration and often use wet sacking or palm leaves to cover the catch, lower the temperature and so reduce spoilage.
Many larger vessels capable of spending a day or more in fishing operations will benefit from the use of some form of on-board preservation, such as ice or chilled seawater (CSW). This category might include artisanal fishing vessels, such as larger dugout canoes, outboard-motor-powered launches and larger inboard-engine-powered vessels up to 20 m long.
With increasing demand for good-quality fresh fish, globalization of the market for these products and increasing awareness of fishermen, the use of ice on board boats is growing. Increase in the use of ice creates a need to ensure that it is used efficiently. Ice production consumes a lot of energy, so unnecessary waste is to be avoided. The most economic way of reducing this waste on board fishing vessels is by using proper storage, such as adequately insulated ice boxes, containers and fish holds where ice is stored and used to preserve the catch.
On small boats portable insulated boxes made of various materials are often used to carry ice to the fishing grounds. Ice is then transferred to the catch in suitable ratios until either all the ice is used, or there is no more space aboard for more fish. Larger boats are able to carry more ice, which allows them to make longer fishing trips, generally with better economic returns for the vessel and crew.
With advances in refrigeration, in particular the advent of compact and relatively lightweight ice-making machines suitable for on-board installation, it is now possible to install ice machines of various types on quite small vessels. This gives a certain measure of independence in fishing operations where trip length is no longer limited by the quantity of ice loaded in port or by how long it will last in the ice hold.
The beneficial effects of using ice can be apparent for a wide range of fishery activities, both small and large scale, and for virtually all species. Ice raises both the quality, and thus the value, of practically all species of fish. This promotes sustainable use of these renewable resources because the harvesting sector is able to preserve catches for longer periods and therefore reduce post-harvest losses.
Most foods are excellent media for rapid growth of microorganisms. There is abundant organic matter in foods, their water content usually sufficient, and the pH is either neutral or slightly acidic.
Foods consumed by man and animals are ideal ecosystems in which bacteria and fungi can multiply. The mere presence of microorganisms in foods in small numbers however, need not be harmful, but their unrestricted growth may render the food unfit for consumption and can result in spoilage or deterioration.
Some organisms grow and elaborate secondary metabolites that may affect the food quality, which may be either desirable or undesirable.. For example, the lactic fermentation of milk is a desired change and is not considered as spoilage, while acetification of wine is an undesirable microbial spoilage. Some organisms may not only cause food spoilage but also produce metabolites which may be extremely toxic to man and animals. Such examples are the production of toxins by clostridia in proteinaceous foods, the elaboration of aflatoxin by aspergilli in feeds etc.
Generally, foods carry a variety of organisms of which, most are saprophytic. Their presence cannot be avoided since these are mostly from the environment in which the food is prepared or processed. Also, their complete elimination is difficult. However, it is possible to reduce their number or decrease their activities by altering the environmental conditions. A knowledge of the factors that either favour or inhibit their growth is therefore, essential in understanding the principles of food spoilage and preservation.
Groups of Food - 1. Highly perishable foods, which ate spoiled rapidly. They include poultry, eggs, meats, most fruits and vegetables and dairy products etc.
2. Semiperishable foods, that spoil less quickly. They include potatoes, apples, nutmeats etc and
3. Nonperishable foods, which are generally kitchen items, like cereals, rice flour, nuts, sugar etc
Initial Contamination of Fresh Foods - Foods consumed by man and animals may be classified into eight main divisions. These are cereal and cereal products, vegetables, fruits, milk and dairy products, meat and poultry, eggs, sea food, and sugar and sugar products. Broadly, these may be considered as plant products and animal products.
Foods may also be classified on the basis of stability :
1. Perishable foods such as meat and fish.
2. Semiperishable foods such as potatoes.
3. Stable foods such as cereals, flour and sugar.
Any stable or semistable food becomes perishable food under moist condition.
Plant products: The internal tissue of whole, healthy plants and fruits are usually free from microorganisms. However, the external surfaces of plant products arc contaminated by microorganisms from the soil, air, insects, human handlers, and packages. Cereals, so long as they are dry, can be preserved for a considerable time, if free from insects.
Sugar products are low in moisture content and possess a hip osmotic pressure, generally adequate to prevent microbial growth. Honey, syrups, etc., are relatively stable, but when diluted with an equal volume of water decompose easily.
Animal products: Animal products are subject to intrinsic as well as environmental and human contamination. The internal portions of a piece of meat are usually free from microorganisms if a healthy animal is properly slaughtered, as in an abattoir by a blow on the head, or by cutting the jugular vein. Meat immediately gets contaminated with microorganisms upon exposure in the abattoir.
The organisms are derived from hides, hair, and intestines of the animals, gloves, hands, and butchering instruments, and the air of the slaughter house. Freshly dressed eviscerated poultry have a microbial flora on their surface. Organisms arc normally present on the live birds, and the manipulations during killing, defeathering, and evisceration introduce organisms on the surface. The microbial flora of freshly caught fish reflects the microbial quality of the water from where they are harvested. Oysters, mussels, and some other shellfish fatten on sewage.
This seafood is potentially capable of transmitting various pathogenic microorganisms. When the fish are cleaned and cut on shipboard under poor handling conditions, they are more likely to be covered by microorganisms, e. g., fish fillets clean, uncracked fresh eggs are usually free from microorganisms with the shell.
The interior of the egg gets contaminated under poor conditions of storage. The shell may be soiled with blood, manures, feathers, nest material or broken eggs. These substances are most likely contaminated with organisms which are drawn in along with the air through the shell pores as the egg cools.
Chemical Changes During Food Spoilage Processes - The biochemical composition of a food, in particular, has a marked influence on the microbial population involved in the spoilage process and the microbial decomposition products associated with the spoilage of that food
Microbes Associated With Food Spoilage - Fruits, vegetables, meats, poultry, sea foods, milk and dairy products and various other food products differ in their biochemical composition and therefore are subject to spoilage by different microbial populations.
Such changes depend upon the nature of the microbes involved in the spoilage. Thus degradation of apple juice by yeast gives an alcoholic taste to the juice. Yeasts convert the carbohydrate into ethanol.
Bacteria which attack food proteins, convert these into .amino acids which are broken down again into foul smelling end products. Digestion of cystein, for example, yields hydrogen sulphide, giving a rotten egg smell to food. Digestion of tryptophan yields indole and skatole which give food a fecal odour.
Influence of Chemical properties of Food on Type of Microbial Growth -
| Chemical properties | Predominant Spoilage organisms |
| Composition: | |
| Protein | Bacteria, moulds |
| Carbohydrate | Yeasts, moulds |
| Fat | Moulds, a few bacteria |
| Acidity: | |
| Acid( | Moulds,Yeasts |
| Nonacid( | Bacteria |
| Osmotic pressure: | |
| Low | Moulds,Bacteria, Yeasts |
| High | Moulds |
Types of Food Spoilage with Causative Organisms -
| Food | Types of spoilage | Causative microorganisms |
| Fresh meat | Putrefaction | Clostridium, Pseudomonas, Porteus, Alcaligenes, Chromobacterium. |
| Cured meat | Mouldy | Penicillium, Aspergillus,Rhizopus. |
| Fish | Discolouration | Pseudomonas |
| Poultry | Odour, Slime | Pseudomonas, Alcaigenes, Xanthomonas. |
| Eggs | Green rot | Pseudomonas Fluorescens |
| Fresh fruits and vegetables | Bacterial soft rot | Erwinia carotovera, Pseudomonas spp. |
| Pickles,Sauer, kraut | Black pickles | Bacillus nigricans |
| Sugar products, Honey, Syrups | Ropy syrup | Aerobacter aerogenes |
| Bread | Mouldy | Rhizopus, Aspergillus |
Microorganisms of Important Food -
| Food Product class | Genera of microbes present in spoiled food during storage |
| Cereal grains (carbohydrates) | Aspergillus,Fusarium, Penicillium ,Monilia,Rhizopus. |
| Bread (carbohydrates) | Bacillus, Aspergillus, Penicillium, Endomyces, Rhizopus, Neurospora. |
| Vegetables(carbohydrates) | Achromobacter, Pseudomonas, Flavobacterium, Lactobacillus, Bacillus. |
| Fruits and juices(carbohydrates) | Acetobacter, Lactobacillus, Saccharomyces,Torulopsis. |
| Fresh meat(protein and lipid) | Micrococcus, Cladosporium, Thamnidium, Achromobacter, Pseudomonas, Flavobacterium. |
| Sausage, bacon, ham etc.(protein and lipid) | Micrococcus, Lactobacillus, Streptococcus, Debarmyces, Penicillium |
| Poultry(protein and lipid) | Achromobacter, Pseudomonas, Flavobacterium, Micrococcus, Salmonella. |
| Fish, Shrimp(protein) | Achromobacter, Pseudomonas, Flavobacterium, Micrococcus, vibrio |
| Milk and milk products (carbohydrates, lipid and protein) | Streptococcus, Lactobacillus, Microbacterium, Achromobacter, Pseudomonas, Flavobacterium, Bacillus |
| Eggs(protein and lipid) | Pseudomonas, Cladosporium, Penicillium, Sporotrichum. |
Factors Affecting Microbial Growth in Food - A variety of factors such as the pH, moisture content, oxidation reduction potential, nutrients, etc., influence microbial activity in foods. For example, each organism has an optimum pH for growth. In general, yeast and fungi are more acid tolerant than bacteria.
Most yeasts favour a pH around 4-4.5 while fungi can tolerate a pH much below, that. Most bacteria favour a pH around 7.0. Thus, both the growth as well as their survival in foods depends on the pH of the food material. The pH of foods varies; some may be neutral while others may be acidic.
Acid foods (pH below 4.5) are not readily spoiled by bacteria but are susceptible to spoilage by yeast and molds. Foods may have a low pH either because of inherent acidity as in fruits and soft drinks or develop acidity as a result of microbial activity as in lactic acid fermentation.
Microorganisms have an absolute, demand for water and the optimum level, of moisture required for growth varies with, the organisms. The water requirement is expressed in terms of available water or water/activity (aw), which is the vapour pressure of the solution divided by the vapour pressure of the solvent. This is equal to the vapour pressure of the solutions in water divided by vapour pressure of the water.
The aw for pure water is 1.00. Each organism has a maximal, optimal and a minimal aw for growth. Most bacteria grow well in a medium of aw activity around 0.995 to 0.998. Molds differ considerably in the optimal aw. For example, Rhizopus sp., has an optimal aw of 0.995-0.980, while Penicillium sp., and has an optimal aw of 0.9935. Also, each mold has an optimal as well as a range of aw for growth.
The aw value of a food is affected by the vapour pressure of solutes such as sugars, salts, hydrophilic colloids or gels. An increase in the concentration of sugars and salts allows the water to be tied up and also causes the removal of water from the microbial cells, The aw value of the food therefore, determines to considerable extent the type of organism that can grow in it.
The oxygen tension or partial pressure of oxygen and the reducing and oxidizing power of the food (O-R potential) influences the growth of organisms. In relation to oxygen, bacteria can be aerobic, anaerobic or facultative, while fungi are mostly aerobic. Yeast are aerobic or facultatively anaerobic. A high O-R potential favours the growth of aerobic and facultative organisms.
Sometimes growth of an aerobe may reduce the O-R potential of food to restrain the growth of other organisms. Most fresh animal and plant foods have a low O-R potential in their interior but have a higher O-R outside. Thus, a fresh piece of meat could support the growth of aerobic organisms in the exterior and the growth of anaerobic organisms inside.
Nutrients in food, their kind and proportions determine the type of organism that will grow. Also, microorganisms vary in their ability to use nutrients. The presence of easily utilizable nutrients will encourage faster growth and quicker damage. For example, a food with easily utilizable sugars will allow better growth than one which contains polysaccharides.
Also, the concentration of the sugars will determine the type and extent of growth, since it affects both the osmotic pressure and the aw- Generally, yeasts and molds are more resistant to high concentrations of sugar than bacteria.
Most foods contain enough peptides and amino acids that they can meet the nitrogen requirement of most organisms found in foods. Some organisms are also proteolytic and can grow on proteins found in the food. The mineral requirement of microorganisms is generally met by the food and this is not a limiting factor.
Some foods may contain antibacterial substances which may prevent bacterial growth and food spoilage. For example, egg is rich in lysozyme and thus, even if the bacteria cross the outer shell (If the egg, they are destroyed by the lysozyme before they can cause any damage.
Minimum Permitting Growth of Microorganisms -
| Group | Minimum aw |
| Bacteria | 0.91 |
| Yeasts | 0.88 |
| Molds | 0.80 |
| Halophilic bacteria | 0.75 |
| Xerophilic fungi | 0.65 |
| Osmophilic yeasts | 0.60 |
Importance of Microbes in Foods - Molds, yeasts and bacteria are important in food spoilage. Which it is true that molds are involved in the spoilage of many foods, some are important in food manufacture specially in mold ripened cheese and the preparation of oriental foods. Various fungi such as species of Aspergillus, Fusarium, and Penicillium have been found in foods and some have been implicated in toxin production.
Yeasts are both useful as well as problem organisms and this depends upon the food. For example, the production of wine is dependent on the growth and activity of the yeast Saccharomyces cerevisiae, while wine can be oxidized to CO2 and water by another wild yeast. Thus, while one yeast is important, the other is a problem.
Important yeasts in foods include species of Saccharomyces which are useful in fermentations, species of Zygosaccharomyces that are osmophilic, are involved in the spoilage of materials such as honey and species of Pichia which form pellicles in liquids such as in beer and wines. Table 17.2 lists some of the yeasts that are important in food spoilage. A variety of bacteria are found in foods and the important ones are grouped based on their biochemical properties
Contamination of Sea Foods
The flora of living fish depends on the microbial content of the waters in which they live. The slime that covers the outer surface of fish has been found to contain bacteria of the genera Pseudomonas, Acinetobacter, Moraxella, Alcal-igenes, Micrococcus*, Flavobacterium, Corynebacterium, Sarcina, Serratia, Vibrio, arid Bacillus. The bacteria on fish from northern waters are mostly psy-chrophiles, whereas fish from tropical waters carry more mesophiles. Freshwater fish carry freshwater bacteria, which include members of most genera found in salt water plus species of Aeromonas, Lactobacillus, Brevibacterium, Alcaligenes, and Streptococcus. In the intestines offish from both sources are found bacteria of the genera Alcaligenes, Pseudomonas, Flavobacterium, Vibrio, Bacillus, Clostridium, and Escherichia. Boats, boxes, bins, fish houses, and fishers soon become heavily contaminated with these bacteria and transfer them to the fish during cleaning. The numbers of bacteria in slime and on the skin of newly caught ocean fish may be as low as 100 and as high as several million per square centimeter, and the intestinal fluid may contain from 1,000 to 100 million per milliliter. Gill tissue may harbor 1,000 to 1 million per gram. Washing reduces the surface count.
Oysters and other shellfish that pass large amounts of water through their bodies pick up soil and water microorganisms in this way, including pathogens if they are present. Alcaligenes, Flavobacterium, Moraxella, Acinetobacter, and some gram-positive bacteria will be found.
Shrimps, crabs, lobsters, and similar seafood have a bacteria-laden slime on their surfaces that probably resembles that offish. Species of Bacillus, Micrococcus, Pseudomonas, Acinetobacter, Moraxella, Flavobacterium, Alcaligenes, and Proteus have been found on shrimp. it°*
The numbers of microorganisms on the skin of fish can be influenced by the method of catching. For example, trawling fish nets along the bottom for long periods results in exposure of the fish to high bacterial counts in the disturbed bottom sediment, and this can be reflected in the initial microbial load on the fish.
Fish cakes and fish sticks or similar products represent a large percentage of the consumed seafood in the
Experiment
ISOLATION AND IDENTIFICATION OF SALMONELLA AND SHIGELLA FROM FOOD SAMPLES
Foods being rich in nutrients carry many microbes. The nature of organism varies with the type of the food, nature of handling, conditions of storage etc. Hence it can transmit many pathogens, one such pathogen is salmonella species , which is the causative organism for salmonellosis. This bacterium is a gram negative, motile, non spore forming rods which come under the family Enterobacteriaceae. This bacterium is a facultative anaerobe and a non lactose fermenter. The initial source of the bacterium is the intestinal tracts of birds and other animals. Human acquire the bacteria from contaminated foods such as beef products, poultry, eggs and egg products.
AIM: To isolate and identify Salmonella species from food.
REAGENTS AND MEDIA:
Mac Conkey agar with (0.15%), bile salt, and Nacl (M081), Salmonella Shigella agar (M108), Xylose deoxycholate agar (M031).
Sample: a) raw fish
EQUIPMENTS:
Petri plates, inoculation loop, Erlenmeyer’s flask, butter paper, pan balance, test tubes.
COMPOSITION AND PREPARATION OF MEDIA:
The composition and preparation of the differential media is given below:
MAC CONKEY AGAR WITH 0.15% BILESALT, CV AND Nacl (M081):
| INGREDIENTS | GRAM/LITRE |
| Pancreatic digest of gelatin Tryptone Lactose Peptic digest of animal tissue Bile mixture Nacl Agar Neutral red Crystal violet pH(at25°C)7.1±0.2 | 17.0 1.5 10.0 1.5 1.5 5.0 15.0 0.030 0.001 |
Suspend 5.15g in 100ml ofdistilled water. Boil to dissolve the medium. Sterilize by autoclaving at 15 lbs pressurefor 15 minutes. Cool to 50°C and pour into sterile Petri plates.
SALMONELLA – SHIGELLA AGAR (M108):
| INGREDIENTS | GRAM/LITRE |
| Beef extract Peptone Lactose Bile salt Sodium citrate Sodium thiosulphate Ferric citrate Brilliant green Neutral red Agar pH(at25°C)7.0±0.2 | 5.0 5.0 10.0 8.5 10.0 8.5 1.0 0.00033 0.025 15.0 |
Suspend 6,3g in 100ml distilled water. Heat to boil with frequent agitation to dissolve the agar completely. Do not auto clave. Cool to 50°C and pour into Petri plates.
XYLOSE LYSINE DEOXYCHOLATE (M031):
| INGREDIENTS | GRAM/LITRE |
| Xylose L-lysine Lactose Sucrose Nacl Yeast extract Sodium deoxycholate Sodium thiosulphate Ferric ammonium citrate Phenol red Agar pH(at25°C)7.0±0.2 | 3.5 5.0 7.5 7.5 5.0 3.0 2.5 6.8 0.8 0.08 15.0 |
Suspend 5.6g in 100ml distilled water. Boil it to dissolve completely. Do not autoclave. Cool to 50°C and pour into sterile plate.
PRINCIPLE:
MAC CONKEY AGAR (M081)
This medium is prepared for the detection, isolation and enumeration of coliforms, intestinal pathogens in water, dairy products and pharmaceutical preparation.Due to the inclusion of specially prepared fraction of bile salts and crystal violet, the medium gives improved differentiation between lactose fermenters and lactose non fermenters. While gram positive cocci are completely inhibited, E.Coli is identified by red to pink colonies andSalmonella by colourless colonies. Shigella is also differentiated by colourless colonies while Staphylococcus species is inhibited.
SS AGAR (M108)
SS agar is a selective medium used for isolation ofSalmonella and Shigella species. It is essentially a modification of the Deoxycholatecitrate agar, described by Leifson. A bile salts mixture replaces deoxycholate for inhibition of coliforms organisms and gram positive bacteria. E.coli is identified by pink to red colonies (poor growth), salmonella, Shigella are identified by colourless colonies.
XLD (XYLOSE LYSINE DEOXYCHOLATE) AGAR (M031):
XLD agar is a selective medium recommended for the isolation of enteric pathogens especially Shigella species. This medium is the first to identifythe following with biochemical crietieria.
- Fermentation of xylose, lactose and sucrose detected by a yellow colour in the presence of phenol red (acid pH).
- Lysine decarboxylation producingred colour(alkaline pH).
- H2S production in a medium i.e not too acid.
Cultural characterisitics after 18-24 hours at 35°C are given below:
| S.No | Organism | Growth | Colour | |
| 1. 2. 3. 4. 5. 6. 7. | E.coli Salmonella enteritis Salmonella typhosa Salmonella paratyphi B Salmonella paratyphi A Shigella flexneri Shigella sonnei | Poor Good –luxuriant Good- luxuriant Good – luxuriant Good Good Good | Yellow Red with black centered Red with black centered Black centered red Red colour Red colour Red colour | |
PROCEDURE:
- Pour the medium into the sterile plates under aseptic conditions.
- Streak the sample onto the medium under aseptic conditions.
- Incubate at room temperature for 24-48 hours.
- Record the observations.
Serial Dilution
Principle: Studies involving the analysis of material such as food, soil, etc require quantitative enumeration of micro organisms. To get countable colony, the sample is tested using standard volume of distilled water. This is then inoculated on the agar medium using pour plate technique and incubated for 24 to 48 hours. Petri dishes having countable colonies ranging from 30 to 300 were chosen for enumeration.
In this experiment, the sample was taken and serially diluted with distilled water.
Material required:
Petri plates, test tubes, test tube stands, cotton, nutrient agar medium, measuring jar and spirit lamp.
Methodology:
Ø Pipettes and Petri plates were sterilized and kept ready for the experiment.
Ø Nutrient agar medium was prepared and sterilized.
Ø Test tube containing 9 ml of distilled water was plugged with cotton, covered with paper and sterilized.
Ø After sterilization, the above materials were cooled and used.
Serial Dilution Procedure:
Ø About 1 ml of the given sample was measured and added to 9 ml of sterile diluted water {10-1}
Ø This was then homogenized using mechanical shaker for two minutes.
Ø 1 ml of the sample from 10-1 was pipetted out into the test tube containing 9 ml sterile diluent using sterile pipette {10-2}
Ø This procedure was repeated using sterile pipette for each dilutions upto 10-9 dilution.
Ø From 10-2, 10-4, 10-6 dilutions, one ml was pipetted out into the labeled Petri plates and also in duplicates.
Ø In each Petri plates 15 to 20 ml of the molten and cooled agar medium was poured under aseptic conditions. Before solidification of the medium, the plates were rotated in clockwise and anti clockwise direction for thorough mixture of the innoculum.
Ø After solidification, the plates were inverted and incubated at 37C [in an incubator] for 24 to 48 hours.
Ø After incubation, the bacterial colonies were counted and recorded.
BIOCHEMICAL TESTS:
The following biochemical tests were performed to prove that the isolate was salmonella species
1. Motility test
2. Indole production
3. Citrate utilization
4. Oxidase test
5. Decarboxylase
6. Urease test
Motility test, indole production, decarboxylase test were done using Motility indole lysine medium (M847). Citrate utilization testusing Simmon’s citrate medium. Urease test using urea broth base and oxidase test using oxidase discs.
- TEST FOR MOTILITY, INDOLE PRODUCTION, DECARBOXYLASE
Aim: To test for motlity, indole production and decarboxylase for the isolate.
PRINCIPLE:
Motility indole lysine medium (M847) is used as an aid for the identification of members of enterobacteriaceae on the basis of motility, indole production and lysine decarboxylase. Motility is indicated by diffused growth while non-motile culture grows along stab line. Lysine decarboxylationshows a purple colour through out the medium. When 3-4 drops of Kovac’s reagent is added to the medium, a pink to red coloured ring indicates a positive reaction.
| COMPOSITION | GRAM/LITRE |
| Peptic digest of animal tissue Casein enzymic hydrolysate Yeast extract L-lysine hydrochloride Dextrose Ferric ammonium citrate Bromo cresol purple Agar pH(at 25°C)6.6±0.2 | 10.0 10.0 3.0 10.0 1.0 0.5 0.02 2.0 |
2. CITRATE UTILIZATION TEST:
AIM: To test for the citrate utilization of the isolate using Simmon’s citrate medium (M099).
PRINCIPLE:
Simmon’s citrate agar medium is recommended for the differentiation of gram enteric bacilli on the basis of citrate utilization. Citrate utilization is indicated by growth and colour change of medium from green to bright blue. Negative reaction means no growth and colour change.
| COMPOSITION | GRAM/LITRE |
| Magnesium sulphate Mono ammonium phosphate Di potassium phosphate Sodium citrate Nacl Agar Bromothymol blue pH(at25°C)6.8 ± 0.2 | 0.2 1.0 1.0 200 5.0 15.0 0.08 |
3. UREASE TEST:
AIM: To test for the presence of urease for isolate.
PRINCIPLE:
Urea broth base is recommended for the identification of the bacteria on the basis of urea utilization especially for the differentiation of Proteus species from Salmonella and Shigella species. Urea broth becomes alkaline, as the urea utilization by the organisms liberate ammonia during the incubation, indicated by pink red colour.
| COMPOSITION | GRAM/LITRE |
| Mono sodium phosphate Di potassium phosphate Yeast extract Phenol red Urea | 9.1 9.5 0.1 0.1 50ml solution |
4. OXIDASE TEST:
AIM: To test the presence of oxidase in the isolate.
PRINCIPLE:
Oxidase disc are used for oxidase test. Oxidase test is carried out by tuching and spreading the well isolate culture on the oxidase disc. The reaction is observed within 2 minutes at 25°C - 30°C. In positive reaction enzyme cytochrome oxidase combines with N,N dimethyl-P-phenylene diamine oxalate and α naphthol to form they dye indo phenol blue, which turns the disc into deep purple colour.
Gram Staining
Principle: the gram stain, a differential stain was developed by Dr. Hans Christian Gram. It is a very useful stain for identifying and classifying the micro organisms in to two major groups: gram positive and gram negative.
In this process, the fixed microbial smear is subjected to four different reagents in the order listed; crystal violet [primary stain], iodine solution, alcohol [decolourising agent] and saffranin [counter stain]. The colour changes occur in the microbial cells at each stage. The micro organism which accepts the primary stain [appears dark blue or violet] are called “gram positive” whereas those that lose the crystal violet and take up the counter stain by saffranin are referred to as “gram negative”. The difference in the staining responses to the gram stain can be related to chemical and physical differences in their cell walls.
The gram negative cell wall is thin, complex, multi layered structure and contains relatively high lipid content, in addition to protein and mucopeptides. The higher amount of lipid is readily dissolved by alcohol, resulting in the formation of large pores in the cell wall which do not close appreciably on dehydration of cell wall proteins, thus facilitating the leakage of crystal – violet – Iodine [CV – I] complex and resulting in the decolorization of the bacterium which later takes up the counter stain and appears red. In contrast, the gram positive cell walls are thick and chemically simple, composed mainly of proteins and cross linked mucopeptides. When treated with alcohol it causes dehydration and closure of cell wall pores, thereby not allowing the loss of crystal – violet – Iodine [CV – I] complex and the cells remain purple.
Materials Required:
24 hour cultures, wash bottles of distilled water, droppers, inoculating tube, glass slides, Bunsen burner or spirit lamp and microscope.
Gram staining reagent:
Ø Crystal violet
Ø Gram iodine solution
Ø 95% ethyl alcohol
Ø Saffranin
Procedure:
Ø Thin smear of the micro organism was made on a separate glass slide and the smear was heat fixed.
Ø The smear was immersed in crystal violet for 30 seconds and washed off with distilled water for a few seconds.
Ø The smear was then immersed in iodine solution for 30 seconds and washed off with 95% ethyl alcohol. The ethyl alcohol was added drop by drop until no more colour flowed from the smear. [at this stage, the gram positive micro organism will not be affected while all gram negative bacteria will be completely de colourised]
Ø The slide was then washed with distilled water
Ø Saffranin [counter stain] was then added to the smear for 30 seconds.
Ø The slide was washed with distilled water and air dried. Finally it was observed under the microscope.
Precautions:
Ø Only fresh young cultures should be used. [Less than 24 hours old] to avoid misleading results.
Ø Excessive heat should be avoided during heat fixing
Ø Over discolourization of the smear should be avoided
Ø Smear should be thin and uniform.
SPOILAGE
Like meat, fish and other seafood may be spoiled by autolysis, oxidation, or bacterial activity or most commonly by combinations of these. Most fish flesh, however, is considered more perishable than meat because of more rapid autolysis by the fish enzymes and because of the less acid reaction of fish flesh that favors microbial growth. Also, many of the unsaturated fish oils seem to be more susceptible to oxidative deterioration than are most animal fats. The experts agree that the bacterial spoilage of fish does not begin until after rigor mortis, when juices are released from the flesh fibers. Therefore, the more this is delayed or protracted, the longer the keeping time of the fish. Rigor mortis is hastened by struggling of the fish, lack of oxygen, and warm temperature and is delayed by a low pH and adequate cooling of the fish. The pH of the fish flesh has an important influence on its perishability, not only because of its effect on rigor mortis but also because of its influence on the growth of bacteria. The lower the pH of the fish flesh, the slower in general bacterial decomposition will be. Lowering of the pH of the fish flesh results from the conversion of muscle glycogen to lactic acid.
FACTORS INFLUENCING KIND AND RATE OF SPOILAGE
The kind and rate of spoilage of fish vary with a number of factors:
1 The kind of fish. The various kinds of fish differ considerably in their perishability. Thus some flat fish spoil more readily than round fish because they pass through rigor mortis more rapidly, but a flat fish like the halibut keeps longer because of the low pH (5.5) of its flesh. Certain fatty fish deteriorate rapidly because of oxidation of the unsaturated fats of their oils. Fishes high in trimethylamine oxide soon yield appreciable amounts of the "stale-fishy" tri-methylamine.
2 The condition of the fish when caught. Fish that are exhausted as the result of struggling, lack of oxygen, and excessive handling spoil more rapidly than those brought in with less ado, probably because of the exhaustion of glycogen and hence smaller drop in pH of the flesh. "Feedy" fish, i.e., those full of food when caught, are more perishable than those with an empty intestinal tract.
3 The kind and extent of contamination of the fish flesh with bacteria. These may come from mud, water, handlers, and the exterior slime and intestinal content of the fish and are supposed to enter the gills of the fish, from which they pass through the vascular system and thus invade the flesh, or to penetrate the intestinal tract and thus enter the body cavity. Even then, growth probably is localized for the most part, but the products of bacterial decomposition penetrate the flesh fairly rapidly by diffusion. In general, the greater the load of bacteria on the fish, the more rapid the spoilage. This contamination may take place in the net (mud), in the fishing boat, on the docks, or, later, in the plants. Fish in the round, i.e., not gutted, have not had the flesh contaminated with intestinal organisms, but it may become odorous because of decay of food in the gut and diffusion of decomposition products into the flesh. This process is hastened by the digestive enzymes attacking and perforating the gut wall and the belly wall and viscera, which in themselves have a high rate of autolysis.
Gutting the fish on the boat spreads intestinal and surface-slime bacteria over the flesh, but thorough washing will remove most of the organisms and adequate chilling will inhibit the growth of those left. Any damage to skin or mucous membranes will harm the keeping quality of the product.
4 Temperature. Chilling the fish is the most commonly used method for preventing or delaying bacterial growth and hence spoilage until the fish is used or is otherwise processed. The cooling should be as rapid as possible 0 to -1 C, and this low temperature should be maintained. Obviously, the warmer the temperature, the shorter the storage life of the fish. Prompt and rapid freezing of the fish is still more effective in its preservation.
5 Use of an antibiotic ice or dip.
EVIDENCES OF SPOILAGE
Since the change is gradual from a fresh condition to staleness and then to inedibility, it is difficult to determine or agree on the first appearance of spoilage. A practical test to determine the quality of fish has been sought for many years, but none has proved entirely satisfactory. A chemical test for trimethylamine is backed by most workers for use on saltwater fish, although some support other methods, such as an estimate of volatile acids or volatile bases or a test for pH, hydrogen sulfide, ammonia, etc. Bacteriological tests are too slow to be useful.
Reay and Shewan (1949) described the succession of external changes in a fish as it spoils and finally becomes putrid. The bright characteristic colors of the fish fade, and dirty, yellow, or brown discolorations appear. The slime on the skin of the fish increases, especially at the flaps and gills. The eyes gradually sink and shrink, the pupil becoming cloudy and the cornea opaque. The gills turn a light pink and finally grayish-yellow color. Most marked is the softening of the flesh, so that it exudes juice when squeezed and becomes easily indented by the fingers. The flesh is easily stripped from along the backbone, where a reddish-brown discoloration develops toward the tail and is a result of the oxidation of hemoglobin.
Meanwhile a sequence of odors is evolved: first the normal, fresh, sea weedy odor, then a sickly sweet one, then a stale-fishy odor due to trimethylamine, followed by ammoniacal and final putrid odors due to hydrogen sulfide, indole, and other malodorous compounds. Fatty fish also may show rancid odors. Cooking will bring out the odors more strongly.
BACTERIA CAUSING SPOILAGE
The bacteria most often involved in the spoilage of fish are part of the natural flora of the external slime of fishes and their intestinal contents. The predominant kinds of bacteria causing spoilage vary with the temperatures at which the fish are held, but at the chilling temperatures usually employed, species of Pseudomonades are most likely to predominate, with Acinetobacter, Moraxella, and Flavobacterium species next in order of importance. Appearing less often, and then at higher temperatures, are bacteria of the genera Micrococcus and Bacillus. Reports in the literature list other genera as having species involved in fish spoilage, such as Escherichia, Proteus, Serratia, Sarcina, and Clostridium. Most of these would grow only at ordinary atmospheric temperatures and probably would do little at chilling temperatures.
Normally pseudomonads increase in numbers on chilled fish during holding, achromobacters* decrease, and flavobacteria increase temporarily and then decrease. The bacteria grow first on the surfaces and later penetrate the flesh. Fish have a high content of nonprotein nitrogen, and autolytic changes caused by their enzymes increase the supply of nitrogenous foods (e.g., amino acids and amines) and glucose for bacterial growth. From these compounds the bacteria make trimethylamine, ammonia, amines (e.g., putrescine and cadaverine), lower fatty acids, and aldehydes, and eventually hydrogen and other sulfides, mercaptans, and indole, which products are indicative of putrefaction. A musty or muddy odor and taste of fish has been attributed to the growth of Strepto-myces species in the mud at the bottom of the body of water and the absorption of the flavor by the fish.
As has been indicated, discolorations of the fish flesh may occur during spoilage; yellow to greenish-yellow colors caused by Pseudomonas fluorescens,yellow micrococci, and others; red or pink colors from growth of Sarcina, Micrococcus, or Bacillus species or by molds or yeasts; and a chocolate-brown color by an asporogenous yeast. Pathogens parasitizing the fish may produce discolorations or lesions.
SPOILAGE OF SPECIAL KINDS OF FISH AND SEAFOODS
The previous discussion has been limited for the most part to the spoilage of fish preserved by chilling. Salt fish are spoiled by salt-tolerant or halophilii bacteria of the genera Serratia, Micrococcus, Bacillus, Alcaligenes, Pseudo monas, and others, which often cause discolorations, a red color being common. Molds are the chief spoilage organisms on smoked fish. Marinated (sour pickled) fish should present no spoilage problems unless the acid content is low enough to permit growth of lactic acid bacteria or the entrance of air per mits mold growth. Frozen fish, too, should present no bacteriological problems after freezing, but of course their quality depends on what has happened to the fish before freezing. Japanese fish sausage is subject to souring caused by volatile acid production by bacilli or to putrefaction, despite the addition of nitrite and permitted preservatives.
In general, shellfish are subject to types of microbial spoilage similar to those for fish. However, in chilled shrimp Acinetobacter, Moraxella; and Vibrio me chiefly responsible for spoilage, although there may be a temporary increase in pseudomonads and a decrease in Flavobacterium. Micrococcus*, and limilIns. Crabmeat is deteriorated by Pseudomonas, Acinetobacter, and Moraxella mi chilling temperatures and mainly by Proteus at higher temperatures. Species of Pseudomonas, Alcaligenes, Flavobacterium, and Bacillus have been incrim-Innicd in the spoilage of raw lobsters. Crabs and oysters may contain species ol Vibrio, including V. parahaemolyticus. The levels in these products fluctuate with seasonal temperature changes.
Oysters remain in good condition as long as they are kept alive in the shell hi i hilling temperature, but they decompose rapidly when they are dead, as in (mucked oysters. The type of spoilage of the shucked oysters depends on the temperature at which they are stored. Oysters are not only high in protein but they contain sugars, which result from the hydrolysis of glycogen. At temper-Mimes near freezing, Pseudomonas, Acinetobacter, and Moraxella species are the most important spoilage bacteria, but Flavobacterium and Micrococcus* species also may grow. The spoilage is termed "souring" although the changes in are chiefly proteolytic. At higher temperatures the souring may be the result of the fermentation of the sugars by coliform bacteria, streptococci, lactobacilli, it iu I yeast to produce acids and a sour odor. Early growth of Serratia, Pseudomonas, Proteus, and Clostridium may take place. An uncommon type of spoilage by an asporogenous yeast causes pink oysters.
Salmonellosis
Salmonellosis may result following the ingestion of viable cells of a member of the genus Salmonella. It is the most frequently occurring bacterial food infection and in some years it is the most frequently occurring bacterial food-borne illness. In addition to the typical food poisoning salmonellosis syndrome, two other disease syndromes can result following consumption of salmonella, and they are compared Classification of the genus Salmonella is confusing, and the naming of organisms does not follow the usual rules of nomenclature. Historically, the names given to isolated salmonellae were related to their pathogenicity in people or animals— for example, S. typhimurium, responsible for typhoid in mice, and S. typhi, responsible for human typhoid. This approach gave way to naming based on the site or location of first isolation in human infections, for example, 5. london, S. panama, and S. Stanleyville. Salmonella isolates are currently identified using the Kauffman-White scheme, a serological procedure in which organisms can be represented by the numbers and letters of the different antigenic sites: O (somatic), Vi (capsular), and H (flagellar). This scheme only identifies antigens of diagnostic importance and does not provide a complete antigenic record of an isolate. The term serovar is used to distinguish strains of different antigenic complements. Further subdivision can be made to biovars, i.e., different sugar fermentation patterns shown by members of the same serovar. This detail is helpful in epidemiological investigations because serovar and biovars can bemused as "markers" to trace the actual route of an outbreak to its source.
The Salmonella infections that are called food poisoning may be caused by any of a large number of serovars (Table 24-7). Usually the infecting bacterium has grown in the food to attain high numbers, increasing the likelihood of infection and often resulting in outbreaks in families or larger groups. By contrast, other intestinal pathogens, such as organisms causing the dysenteries and typhoid and paratyphoid fevers, usually have a longer incubation period before symptoms and, except under epidemic conditions, occur in only scattered cases.
The Organism salmonellae ferment glucose, usually with gas, usually do not ferment lactose or sucrose. Like other bacteria, they will grow over a wider range of temperature, pH, and aw in a good culture medium rather than in a poor one. For example, minimal temperatures for growth in foods range from 6.7 to 7.8 C in chicken a la king to over 10 C in custard. Depending on the food and the serotype, the £>& c values range from 0.06 to 11.3 min. Recommendations for thermal destruction of salmonellae in perishable foods are similar to those for staphylococci, namely, heating to 66_C_and holding all parts at that temperature for at least 12 min .F140 values (minutes at 140 F necessary to reduce an inoculums to an undetectable level) found for two species were 78_and 19 min, respectively
The likelihood of infection by consumption of a food containing salmonellae depends on the resistance of the consumer, the infectiveness of the particular strain of Salmonella, and the number of organisms ingested. Less infective species such as S. pullorum must be ingested in hundreds of millions or in billions to bring about infection, but considerably fewer (about a million) organisms of more infective species, e.g., S. enteritidis, usually would be sufficient. Salmonellae apparently can attain considerable numbers in foods without causing detectable alterations in appearance, odor, or even taste. Of course, the more of any of these pathogens the food contains, the greater the likelihood of infection on the person who eats the food and the shorter the incubation time.
Sources of Salmonella Human beings and animals are directly or indirectly the source of the contamination of foods with salmonellae. The organisms may come from actual cases of the disease or from carriers. Most frequently isolated serovars, such as S. typhimurium and others cause human gastroenteritis, but any of many other types may be responsible. The organisms also may come from cats, dogs, swine, and cattle, but more important sources for foods are poultry and their eggs and rodents. Chickens, turkeys, ducks, and geese may be infected with any of a large number of types of Salmonella, which are then found in the fecal matter, in eggs from the hens, and in the flesh of the dressed fowl. About one-third of all the food products involved in Salmonella outbreaks are meat and poultry products. Considerable attention has been given to shell eggs and to liquid, frozen, and dried eggs as sources of Salmonella. Infected rodents, rats and mice, may contaminate unprotected foods with their feces and thus spread Salmonella bacteria. Flies may play an important role in the spread of Salmonella, especially from contaminated fecal matter to foods. Roaches apparently also can spread the disease.
Changes in processing, packaging, and compounding of foods and feeds in recent years have resulted in an apparent increase in salmonellosis from these products. Salmonellae have been introduced by the incorporation of cracked and dried eggs in baked goods, candy, ice cream, and convenience foods such as cake and cookie mixes. The compounding of new food products may make possible the growth of salmonellae or other food-poisoning organisms, introduced by means of an ingredient in which they had been unable to grow, or these organisms may be in a product when sold and become able to grow in this food as it is modified for use. Large-scale handling of foods, as by commissaries or institutions, tends to increase the spread of trouble, and food vending machines add to the risk, as do precooked foods.
Feeds, especially those from meat or fish by-products, may carry salmonellae to poultry or meat animals. Even pet feeds have been known to transmit salmonellae to domestic animals, from which children have been infected.
Food Involved A large variety of foods are involved in causing outbreaks of Salmonella infections. Most commonly incriminated are various kinds of meats, poultry and products from them, especially if they are held unrefrigerated for long periods. Fresh meats may carry Salmonella bacteria that caused disease in the slaughtered animals or may be contaminated by handlers. Meat products, such as meat pies, hash, sausages, cured meats (ham, bacon, and tongue), sandwiches, and chili, often are allowed to stand at room temperatures, permitting the growth of salmonellae. Poultry and its dressing and gravy should not give trouble if properly handled and cooked but often are mishandled, as are fish and other seafood and products from them. Milk and milk products, including fresh milk, fermented milks, ice cream, and cheese, have caused infections. Since eggs may carry the salmonellae, foods made with eggs and not sufficiently cooked or pasteurized may carry live organisms, e.g., pastries filled with cream or custard, cream cakes, baked
The Disease As with other infectious diseases, individuals differ in their susceptibility to Salmonella infections, but in general morbidity is high in any outbreak. As has been stated, the susceptibility of humans varies with the species and strain of the organism and the total numbers of bacteria ingested.
A longer incubation period usually distinguishes salmonellosis from staphylococcus poisoning: usually 12 to 36.hr for the former and about 2 to 4 hr for the latter. Shorter (as little as 5 hr) or longer (up to 72 hr) incubation periods may occur in some cases of Salmonella infections.
The principal symptoms of a Salmonella gastrointestinal infection are nausea and vomiting appear suddenly. This may be preceded by a headache and chills. Other evidences of the disease are watery, greenish foul-smelling stools, prostration, muscular weakness and faintness. Mortality is low, being less than 1 %. The severity and duration vary not only with the amount of food eaten and hence the numbers of Salmonella bacteria ingested but also with the individual. Intensity may vary from slight discomfort and diarrhea to death in 2 to 6 days. Usually the symptoms persist for 2 to 3 days, followed by uncomplicated recovery, but they may linger for weeks or months. About 0.2 to 5 percent of the patients may become carriers of the Salmonella organism.
The laboratory diagnosis of the disease is difficult unless Salmonella can be isolated from the suspected food and from the stools of individuals. Often, however, the incriminated foods are no longer available, and the organisms disappear from the intestinal tract.
Conditions Necessary for an Outbreak The following conditions are necessary for an outbreak of a food-borne Salmonella gastrointestinal infection: (1) The food must contain or become contaminated with the Salmonella bacteria, (2) these bacteria must be there in considerable numbers, either because of contamination or more often because of growth; these high numbers mean that the food must be a good culture medium, the temperature must be favorable, and enough time must be allowed for appreciable growth, and (3) the viable organisms must be ingested.
Prevention of Outbreaks Three main principles are involved in the prevention of outbreaks of food-borne Salmonella infections: (1) avoidance of contamination of the food with salmonellae from sources such as diseased human beings and animals and carriers and ingredients carrying the organisms, e.g., contaminated eggs, (2) destruction of the organisms in foods by heat (or other means) when possible, as by cooking or pasteurization, paying special attention to held-over foods, and (3) prevention of the growth of Salmonella in foods by adequate refrigeration or by other means. In the prevention of contamination, care and cleanliness in food handling and preparation are important. The food handlers should be healthy (and not be carriers) and clean. Rats and other vermin and insects should be kept away from the food. Ingredients used in foods should be free of salmonellae, if possible. Of course foods should not be allowed to stand at room temperature for any length of time, but if this happens, thorough cooking will destroy the Salmonella organisms (but not staphylococcus enterotoxin). Warmed-over leftovers, held without refrigeration, often support the growth of Salmonella, as may canned foods that have been contaminated and held after the cans were opened. Inspection of animals and meats at packing houses may remove some Salmonella infected meats but is not in itself a successful method for the prevention of human salmonellosis
What is shigellosis?
Shigellosis is an infectious disease caused by a group of bacteria called Shigella. Most who are infected with Shigella develop diarrhea, fever, and stomach cramps starting a day or two after they are exposed to the bacterium. The diarrhea is often bloody. Shigellosis usually resolves in 5 to 7 days. In some persons, especially young children and the elderly, the diarrhea can be so severe that the patient needs to be hospitalized. A severe infection with high fever may also be associated with seizures in children less than 2 years old. Some persons who are infected may have no symptoms at all, but may still pass the Shigella bacteria to others.
What sort of germ is Shigella?
The Shigella germ is actually a family of bacteria that can cause diarrhea in humans. They are microscopic living creatures that pass from person to person. Shigella were discovered over 100 years ago by a Japanese scientist named Shiga, for whom they are named. There are several different kinds of Shigella bacteria: Shigella sonnei, also known as "Group D" Shigella, accounts for over two-thirds of the shigellosis in the
How can Shigella infections be diagnosed?
Many different kinds of diseases can cause diarrhea and bloody diarrhea, and the treatment depends on which germ is causing the diarrhea. Determining that Shigella is the cause of the illness depends on laboratory tests that identify Shigella in the stools of an infected person. These tests are sometimes not performed unless the laboratory is instructed specifically to look for the organism. The laboratory can also do special tests to tell which type of Shigella the person has and which antibiotics, if any, would be best to treat it.
How can Shigella infections be treated?
Shigellosis can usually be treated with antibiotics. The antibiotics commonly used for treatment are ampicillin, trimethoprim/sulfamethoxazole (also known as Bactrim* or Septra*), nalidixic acid, or ciprofloxacin. Appropriate treatment kills the Shigella bacteria that might be present in the patient's stools, and shortens the illness. Unfortunately, some Shigella bacteria have become resistant to antibiotics and using antibiotics to treat shigellosis can actually make the germs more resistant in the future. Persons with mild infections will usually recover quickly without antibiotic treatment. Therefore, when many persons in a community are affected by shigellosis, antibiotics are sometimes used selectively to treat only the more severe cases. Antidiarrheal agents such as loperamide (Imodium*) or diphenoxylate with atropine (Lomotil*) are likely to make the illness worse and should be avoided.
Are there long term consequences to a Shigella infection?
Persons with diarrhea usually recover completely, although it may be several months before their bowel habits are entirely normal. About 3% of persons who are infected with one type of Shigella, Shigella flexneri, will later develop pains in their joints, irritation of the eyes, and painful urination. This is called Reiter's syndrome. It can last for months or years, and can lead to chronic arthritis which is difficult to treat. Reiter's syndrome is caused by a reaction to Shigella infection that happens only in people who are genetically predisposed to it.
Once someone has had shigellosis, they are not likely to get infected with that specific type again for at least several years. However, they can still get infected with other types of Shigella.
The Shigella bacteria pass from one infected person to the next. Shigella are present in the diarrheal stools of infected persons while they are sick and for a week or two afterwards. Most Shigella infections are the result of the bacterium passing from stools or soiled fingers of one person to the mouth of another person. This happens when basic hygiene and handwashing habits are inadequate. It is particularly likely to occur among toddlers who are not fully toilet-trained. Family members and playmates of such children are at high risk of becoming infected.
Shigella infections may be acquired from eating contaminated food. Contaminated food may look and smell normal. Food may become contaminated by infected food handlers who forget to wash their hands with soap after using the bathroom. Vegetables can become contaminated if they are harvested from a field with sewage in it. Flies can breed in infected feces and then contaminate food. Shigella infections can also be acquired by drinking or swimming in contaminated water. Water may become contaminated if sewage runs into it, or if someone with shigellosis swims in it.
What can a person do to prevent this illness?
There is no vaccine to prevent shigellosis. However, the spread of Shigella from an infected person to other persons can be stopped by frequent and careful handwashing with soap. Frequent and careful handwashing is important among all age groups. Frequent, supervised handwashing of all children should be followed in day care centers and in homes with children who are not completely toilet-trained (including children in diapers). When possible, young children with a Shigella infection who are still in diapers should not be in contact with uninfected children.
People who have shigellosis should not prepare food or pour water for others until they have been shown to no longer be carrying the Shigella bacterium.
If a child in diapers has shigellosis, everyone who changes the child's diapers should be sure the diapers are disposed of properly in a closed-lid garbage can, and should wash his or her hands carefully with soap and warm water immediately after changing the diapers. After use, the diaper changing area should be wiped down with a disinfectant such as household bleach, Lysol* or bactericidal wipes.
Basic food safety precautions and regular drinking water treatment prevents shigellosis. At swimming beaches, having enough bathrooms near the swimming area helps keep the water from becoming contaminated.
Simple precautions taken while traveling to the developing world can prevent getting shigellosis. Drink only treated or boiled water, and eat only cooked hot foods or fruits you peel yourself. The same precautions prevent traveler's diarrhea in general.
Every year, about 18,000 cases of shigellosis are reported in the
In the developing world, shigellosis is far more common and is present in most communities most of the time.
What else can be done to prevent shigellosis?
It is important for the public health department to know about cases of shigellosis. It is important for clinical laboratories to send isolates of Shigella to the City, County or State Public Health Laboratory so the specific type can be determined and compared to other Shigella. If many cases occur at the same time, it may mean that a restaurant, food or water supply has a problem which needs correction by the public health department. If a number of cases occur in a day-care center, the public health department may need to coordinate efforts to improve handwashing among the staff, children, and their families. When a community-wide outbreak occurs, a community-wide approach to promote handwashing and basic hygiene among children can stop the outbreak. Improvements in hygiene for vegetables and fruit picking and packing may prevent shigellosis caused by contaminated produce.
Some prevention steps occur everyday, without you thinking about it. Making municipal water supplies safe and treating sewage are highly effective prevention measures that have been in place for many years.
Preservation
Fish is the most susceptible to autolysis, oxidation and hydrolysis of fats, and microbial spoilage. Its preservation therefore involves prompt treatment by preservative methods, and often these methods are rigorous compared with those used on meats. When fish are gathered far from the processing plant, preservative methods must be applied even on the fishing boat. Evisceration should be done promptly to stop active digestive enzymes in the gut. Advantages gained by gutting may be offset by a possible delay in rapid cooling of the fish.
Rigor mortis is especially important in the preservation offish, for it retards postmortem autolysis and bacterial decomposition. Therefore, any procedure that lengthens rigor mortis lengthens keeping time. It is longer if the fish have had less muscular activity before death and have not been handled roughly and bruised during catching and later processing, and it is longer in some kinds of fish than in others. The ultimate pH of the flesh after death is related to the amount of glycogen available at death. The more glycogen present, the lower the pH. The less muscular activity before death, the higher the level of glycogen,, or the lower the ultimate pH. Reducing the holding temperature will lengthen the period.
Aseptic methods to reduce the contamination of seafood are difficult to apply, but some of the gross contamination before processing can be avoided by general cleaning and sanitization of boats, decks, holds, bins, or other containers and processing equipment in the plant and by use of ice of good bacteriological quality. The removal of soil from contaminating surfaces and from the fish by adequate cleaning methods, including effective detergent solutions, helps greatly to reduce the microbial load on the fish.
The removal of organisms is difficult, but the fact that most of the contamination is on the outer surface of the fish and other seafood permits the removal of many of the microorganisms by washing off slime and dirt.
USE OF HEAT
Live crabs are cooked in retorts at temperatures up to 121 C to facilitate removing the meat from the shell. Hand picking and hand packing of the meat is common. Processing times and temperatures for canned crabmeat range from 85.6 to 87.2 C for 92 to 150 min (Dickerson and Berry, 1974). These processes are considered pasteurization, and cans are preserved by refrigeration. Some seafoods, e.g., oysters, are "canned" by packing into cans or jars and are not heat-processed but are preserved by refrigeration.
Most canned seafoods, however, are heat-processed so that they are sterile, or at least "commercially sterile." Like meats, seafoods are low-acid foods and for the most part have a slow rate of heat penetration and hence are difficult to heat-process. Also, some kinds of seafoods soften considerably or even fall apart when sterilization in the can is attempted.
The process varies with the product being canned and the size and shape of the container. In general, the heat processes are more severe than those used for meats, but some special products are lightly processed. Canning practices carried out in accordance with the FAO code of practice (FAO, 1973) minimize health hazards arising from canned seafoods.
USE OF LOW TEMPERATURES
It is only after death of the fish or other sea animal that autolysis gets under way, with softening and production of off-flavor, and microbial growth becomes uncontrolled; as has been stated, these changes are delayed by rigor mortis. Oysters in their shells, for example, will not decompose so long as they remain alive, and life is lengthened by chilling storage of shell oysters. Carp seined from mid western lakes have been kept alive and hence in good condition by shipment in tanks to the
Chilling
Because fish flesh autolyzes and the fats become oxidized at temperatures above freezing—rapidly at summer temperatures and more slowly as the temperature is dropped toward freezing—preservation by chilling temperatures is at best temporary. When fish or other seafood is obtained at some distance from the receiving plant, the necessity for chilling on the boat depends on the kind of fish, whether it is dressed there, and the atmospheric temperature. In general, small fish are more perishable than large ones, and dressed fish autolyze more slowly than whole fish but are spoiled more readily by bacteria. When outside temperatures are warm and distances of transportation are great, it becomes necessary to chill the fish and related foods on the fishing boat by packing in crushed ice or by mechanical refrigeration in order to slow autolysis and microbial growth until the products are marketed or are processed for longer preservation. The incorporation of preservatives in the ice used for chilling fish will be discussed subsequently. The time allowable for holding in ice or in chilling storage will vary considerably with the kind of fish or other seafood but will not be long in most instances. In general, chilling storage on shore is useful only when retail markets are near at hand and turnover is rapid. Otherwise, some other method of preservation is applied, such as freezing, salting, drying, i smoking, canning, or combinations of these methods.
Freezing
Most of the modern methods offreezing foods initially were developed for freezing fish. In earlier days, ice with added salt was employed. With the advent of mechanical refrigeration, sharp freezing was employed and the fish were "glazed"; i.e., a layer of ice was frozen around the outside. Whole fish, especially the larger ones, usually are sharp-frozen in air or in a salt brine. Quick freezing is applied to wrapped fillets or steaks, although whole smaller fish may be so frozen. Like meats, quick-frozen fish may thaw to more like their original condition than fish frozen more slowly. During storage the fats of frozen fish are subject to hydrolysis and oxidation. Fatty fish deteriorate more rapidly than lean ones, probably because of more hydrolysis.
Decapitated raw shrimp are frozen and glazed, and some cooked shrimp are rozen. Other seafoods preserved by freezing include scallops, clams, oysters, spiny lbbster tails, and cooked crab and lobster meat. Most of these products are packaged before freezing.
As with meats, freezing kills some but not all of the microorganisms present, and growth will take place after thawing if time permits. Fish carry a flora of psychrotrophic bacteria, most of which survive freezing and are ready to grow on thawing, e.g., Pseudomonas, Acinetobacter, Moraxella, Alcaligenes, and Flavobacterium species. Spores of type E Clostridium botulinum will survive freezing and storage and may grow and produce toxin when temperatures reach 3.3 C or above. Frozen raw seafoods contain few enterococci, coliforms, or
staphylococci. Numbers of these organisms may be increased in the processing plant by cutting, breading, and battering operations. Precooking reduces only coliforms to any extent.
USE OF IRRADIATION
Preservation offish by ultraviolet rays has been tried but not put into practice. Experiments have indicated that gamma or cathode irradiation of some kinds of fish may be successful.
PRESERVATION BY DRYING
The dry-salting of fish or immersion in brine constitutes a method of drying, in that moisture is removed or tied up. Oxidation of fish oils is not retarded and may cause deterioration. Salting of fish is being done to a lesser extent in the
Sun drying of fish, either of small fish or of strips of flesh, is not practiced extensively in the
Part of the preservative effect of smoking is a result of the drying of the fish.
USE of PRESERVATIVES
The salting or marination of fish by dry salt or in brine is effective not only because of the drying effect mentioned in the preceding section but also because of the effect of the sodium chloride as a chemical preservative. This method is used to a considerable extent in many countries. The chemical and bacteriological qualities of the salt are important, for impurities such as calcium and magnesium salts may hinder the penetration of the sodium chloride, and halophilic or salt-tolerant bacteria that are introduced may cause discolor-ations of the fish.
Because of the great perishability of fish, investigators have tried numerous chemicals as preservatives, either applied directly to the fish or incorporated in the ice used in chilling them.
Preservatives Used on Fish
In the intensive search for chemical preservatives that could be applied directly or as dips to round fish or fillets, a large number of chemicals have been tried, ranging from those which most control agencies would approve to those whose use would be questionable. Sodium chloride, an acceptable preservative, has been discussed.
Fish may be dry-salted so as to contain 4 to 5 percent salt. The salt contributes halophiles which may discolor the fish (e.g., a red color from Serratia sal-inaria*). Species of Micrococcus* usually grow on the fish, and there is a decrease in Flavobacterium, Alcaligenes, Pseudomonas, and others. Curing of fish may be "mild," i.e., with light salting, or may be in heavy brine or with solid salt and may be followed by smoking. Benzoic acid and benzoates have been only moderately successful as preservatives. Sodium and potassium nitrites and nitrates have been reported to lengthen the keeping time and are permitted in some countries. Sorbic acid has been found to delay spoilage of smoked or salted fish. Boric acid has been used in Europe with some improvement in the keeping quality, but its use is illegal in the
Antibiotics also have been tried experimentally, usually in a dip or in ice. Of those tested, chlortetracycline and oxytetracycline seemed best, and now their use is permitted. Chloramphenicol is fairly effective, and penicillin, streptomycin, and subtilin are poor or useless.
Storage of fish in an atmosphere containing increased levels of carbon dioxide has been found to lengthen the keeping time. The normal spoilage flora is replaced with lactobacilli and others, and the product "sours" when it begins to spoil.
Pickling offish may mean salting or acidification with vinegar, wine, or sour cream. Herring is treated in various ways: salted, spiced, and acidified. Various combinations of these treatments, coupled with an airtight container, preserve the fish, although refrigeration also must be employed for some products.
Formerly, fish was smoked primarily for its preservation, and the smoking was heavy, but now that canning, chilling, and freezing are available to lengthen keeping time, much of the smoking of fish is primarily for flavor and hence is light. The smoke treatment and other preservative methods combined with it vary with the kind of fish, the size of pieces, and the keeping time desired. Fish to be smoked usually are eviscerated and decapitated but may be in the round, split, or cut into pieces. Commonly, salting, light or heavy, precedes smoking and serves not only to flavor the fish but also to improve its keeping quality by reducing the moisture content. Drying may be aided by air currents. The smoking may be done at comparatively low temperatures (26.7 to 37.8 C) or at high temperatures such as 63 to 92 C which result in partial cooking of the fish.
The principles of preservation involved in smoking fish are similar to those discussed in Chapter 9.
Microbiology of Fish Brines Numbers of bacteria in fish-curing brines vary with the concentration of salt, the temperature of the brine, the kind and amount of contamination from the fish introduced, and the duration of use of the brine and range from 10,000 to 10 million bacteria per milliliter. Salt concentrations usually are between 18 percent and saturation but may be lower, especially after fish are introduced. The higher the temperature of the brine, the more salt
necessary to prevent its spoilage. Contamination comes from the fish, which ordinarily introduce species of Pseudomonas, Acinetobacter, Moraxella, Alcaligenes, and Flavobacterium; from ice, which introduces these genera plus (Orynebacterium and cocci; and from mechanically introduced sources, e.g., dust, which add cocci. On continued use of the brine, numbers of organisms increase, because of addition from successive lots offish and because of growth Of salt-tolerant bacteria such as the micrococci*. As the brine ages, there is a decrease in numbers and an increase chiefly in corynebacteria in low-salt brines and in micrococci* in high-salt brines.
Preservatives Incorporated in Ice
So-called germicidal ices are prepared by adding a chemical preservative to water before freezing. These ices are eutectic when the added chemical is uniformly distributed throughout, as with sodium chloride, or noneutectic when distribution is not uniform, as with sodium benzoate. Noneutectic ice is finely crushed for use on fish so as to get the chemical evenly spread in it.
Many investigators have sought the ideal chemical to be incorporated in ice for icing fish and have tested with some success a large number of chemicals, including hypochlorites, chloramines, benzoic acid and benzoates, colloidal silver, hydrogen peroxide, ozone, sodium nitrite, sulfonamides, antibiotics, propionates, levulinic acid, and many others. Both the American and Canadian governments and those of other nations now permit the incorporation of the tetracyclines at up to 7 ppm in ice to be used by fishers to preserve fish on trawlers and during transportation.
The purpose of the application of preservative chemicals to fish either directly or as dips or germicidal ices is to kill or inhibit microorganisms on the surfaces of the fish, where at first they are most numerous and active.
Antioxidants
Fats and oils of many kinds of fish, especially the fatter ones, such as herring, mackerel, mullet, and salmon, are composed to a great extent of unsaturated fatty acids and hence are subject to oxidative changes, producing oxidative rancidity and sometimes undesirable alterations in color. To counteract these undesirable changes, antioxidants may be applied as dips, coatings, glazes, or gases. Good results have been reported with nordihydroguaiaretic acid, ethyl gallate, ascorbic acid, and other compounds and with storage in carbon dioxide.
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