Cell disruption technique - grinding with sand

 

 Aim

To demonstrate the breaking up of cell structures using abrasives like sand particles.

Principle

 In order to release microbial cell contents during downstream processing, a number of methods can be used for cell disruption to disintegrate the extremely tough cell walls. Any potential method of disruption must ensure that liable materials are not denatured by the process or hydrolyzed by enzymes present in the cell. The use of combination of different techniques release products from specific locations within yeast cell. Although many techniques are available which are satisfactory at laboratory scale, only limited number have been proved to be suitable for large-scale applications, particularly for intracellular enzyme extraction. Types of cell disruption methods are physical, mechanical and chemical.

Grinding with sand is a mechanical cell disruption method used to break microbial, plant, or animal cells to release intracellular components such as enzymes, proteins, nucleic acids, pigments etc. This technique tears up the structures of a cell using abrasive sand particles. Abrasion and shear force produced by grinding cells with fine sand particles mechanically rupture the cell wall and cell membrane. This helps to achieve the release of cytosolic contents, and allows some degree of breakup of physical structures. Physical structures are further removed by isopycnic centrifugation.

Grinding with sand is commonly used for laboratory-scale extraction from microorganisms and plant tissues. Usually, the process is carried out at low temperature to prevent denaturation of cellular components.

 

Materials Required

1.Yeast culture

2.Mortar and pestle

3.Sterile fine Sand

4. Sabouraud's dextrose Agar

5. Routine microbiological facilities

 

 

Procedure

1. Five ml of yeast suspension was taken into sterile test tube. One test tube was kept as control.

2. The yeast suspension was taken in a sterile mortar, and cloth filtered fine sand was added to it.

3. It was then ground with pestle several times and serially diluted.

4. 1 ml of suspension from 10 ^-4 and 10^-5 dilutions were plated on Sabouraud's dextrose agar and 

    CFU was counted

5.1 ml of the suspension from the control tube was also serially diluted and CFU was counted.

 

Result

Sabouraud's dextrose Agar plated with the sample ground with sand particle showed a fall in colony count, which indicate fall in cell count. 

 

Conclusion

Grinding with sand is a mechanical cell disruption technique in which the abrasive action of sand particles breaks the cell wall and membrane, releasing intracellular materials.

 

Production of Wine

 

Aim

To perform fermentation of grape juice into alcohol by the action of yeast cells.

Principle

Wine is traditionally used as an alcoholic beverage which is prepared from fermentation of grape. Other fruits such as apple, pears, plums etc. can also be used for production of wine. Usually, wine is prepared by using the microorganism Saccharomyces cerevisiae.

During wine production, yeast convert glucose and fructose present in the fruit is enzymatically converted first to aldehyde and then to ethyl alcohol. Grapes containing 20-30% sugar concentration will yield wine containing approximately 10 - 15% alcohol. Grapes also contain minerals, tannins, pigments, vitamins, enzymes and other aromatic compounds whose concentration in the final product give characteristic taste. The quality of wine depends on grape variety, fermentation conditions, aging process and microbial activity.

The science of wine production (enology) starts with collection of grapes. The protocol; for wine making include preparation of must. Fermentation is performed by adding microorganism Saccharomyces cerevisiae to the must. It is incubated at room temperature for 14-21 days. Must is collected, transferred to large bottle for settling, clarification and storage. The ageing include storage for along period (1-5 years) in a wooden tank or in other ageing tank. The chemical changes that occur during ageing is responsible for aroma. The final product, wine contain 10 - 15% alcohol.

Grape wine is of two types: red wine and white wine. Red wine comes from red and purple skin varieties of grape whereas white wine is prepared from white skin grapes or from red or purple grapes with skin removed. This experiment is a modified method in which red wine is produced from red grape juice. The fermenting wine is examined at one week interval, during incubation, for 

(1)total acidity (1% tartaric acid)

(2) volatile acidity (1% acetic acid)

(3) aroma - fruity, yeast like, sweet

(4) taste - bitter, sweet, sour

(5) pH 3-3.9

(6) alcohol (expressed in volume percentage).

 Materials Required:

Fresh grapes, Sugar, Yeast, sterile water.

Equipments: 

Burette, conical flask, pipettes, graduated cylinder, test tubes

Reagents:

1 % phenolphthalein in 0.1 N NaOH, potassium dichromate solution and sulphuric acid

 

Procedure:

1.     1 kg of grapes were washed thoroughly and crushed to obtain juice. The juice was filtered and around 750 ml was obtained, which was separated in two flasks as 700 ml and 50 ml.

2.     The flask with 50 ml juice was inoculated with yeast and placed in a mechanical shaker for 48 hrs to develop the inoculum.

3.     The inoculum was then added to the flask with 700 ml juice. 25 ml was withdrawn as the initial sample for evaluating total acidity, taste, flavour and aroma.

4.     The inoculated juice was allowed to undergo fermentation at room temperature (25- 30⁰C) for 21 days. 20 g of glucose was supplemented on the 7^th and 14^th days.

5.     During fermentation, samples were tested for acidity and alcohol content at 7 days intervals.

 Estimation of Parameters

        a)     pH of fermenting wine was tested with the help of pH paper. Aroma and taste were noted. 

        b)    Total Acidity (% of tartaric acid)

To 10 ml aliquot of fermented wine sample, 10 ml of distilled water and 5 drops of 0.1% phenolphthalein solution were added. The contents were mixed and titrated with 0.1 N NaOH, to the first persistent pink colour.

The total acidity was calculated using the formula:

% of tartaric acid = millilitre of alkali x Normality of alkali x 7.5

                                    Weight of sample in gram

1 ml = 1g

c)    Volatile Acidity

    Following titration, volatile acidity was calculated using the formula:

% of acetic acid = millilitre of alkali x Normality of alkali x 6

                                    Weight of sample in gram

1 ml = 1g

d) Estimation of Ethanol

Estimation of Ethanol was performed by adding potassium dichromate reagent and checking the OD at 600 nm

 

Potassium dichromate solution: 34 g of potassium dichromate (K₂Cr₂O₇) was dissolved in 500 ml of distilled water in 1l flask. 32.5 ml conc. H₂SO₄ was added by keeping the flask in ice bucket.

 

Preparation of standard curve for alcohol concentration

1)     1-10 % of alcohol samples were prepared.

2)     1.5 ml each of various concentration alcohol samples were taken in different test tubes.

3)     2.5 ml of potassium dichromate reagent and 1 ml of distilled water were added and test tubes incubated at 600C in water bath for 30 minutes.

4)     Optical density was measured at 600 nm

5)     A standard curve was plotted with concentration of alcohol on x-axis and optical density at 600 nm on Y -axis.

 

Determination of concentration of alcohol of fermented sample

1)     1.5 ml of wine sample was mixed with 2.5 ml of potassium dichromate reagent and 1 ml of distilled water were added and test tubes incubated at 60⁰C in water bath for 30 minutes.

2)     Optical density was measured at 600 nm

  

Dilution	Optical Density
Stock Solution
2 %
4 %
6 %
8 %
10%
Wine Sample
W1
W2

 

Result

After 21 days, % tartaric acid in the fermented wine was noted. pH and alcohol content was also noted.

 

Observation

Fermented Wine
	1st day	7th day	14th day	21st day
% tartaric acid
% acetic acid
pH
Taste
Aroma
% of alcohol

 

 

 

 

Shigella dysenteriae

Dysentery is a clinical condition of multiple origin. It could be bacillary or amoebic in nature. Dysentery is characterised by frequent discharge of blood stained, mucopurulent stools. Infection with this organism often leads to ulceration of the intestinal epithelium.

Shigella  - gram-negative, facultatively anaerobic, rod-shaped, non motile, non-sporing, non capsulated - extremely pathogenic and causes severe dysentery.   Fimbriae may be present.

Shigella produce an exotoxin (Shiga toxin) which disrupts protein synthesis and produces endothelial damage. Infection with this organism often leads to ulceration of the intestinal epithelium. Shigella spread via fecal-oral and person-to-person transmission

Kiyoshi Shiga - Japanese physician and bacteriologist- In 1896, Shiga discovered and identified Shigella dysenteriae which caused dysentery in Japan, and the Shiga toxin which is produced by the bacteria. He conducted research on other diseases such as tuberculosis and trypanosomiasis.

Most individuals are infected with Shigellae when they ingest food or water contaminated with human fecal material. This results in Bacillary dysentery.

Shigella can survive upto 30 days in milk, eggs and cheese.

Bacillary dysentery is characterized by severe abdominal cramps and the frequent painful passage of low volume stools containing blood and pus

MORPHOLOGY

Shigella are short Gram -ve rods-non-sporing, non-motile-non-capsulated. Fimbriae are present only in S. flexneri

CULTURAL CHARACTERISTICS 

Aerobic and facultative anaerobes. Optimum temperature 37°C. They grow on ordinary media however less readily than other Enterobacteria.

Nutrient agar and Blood agar

On Nutrient agar and Blood agar, colony are smooth, circular convex greyish or colorless, translucent often 2-3 mm diameter.

MacConkey agar (MA)

On MA, colonies are pale and yellowish (non-lactose fermenting). 

Exception S. sonnei being late lactose fermenting,  become pink when incubation period is prolonged

Deoxycholate citrate agar (DCA)

DCA - excellent selective medium for isolation of Shigella from faeces. Colonies are pale and similar to though usually slightly smaller 1-1.5mm diameter and more translucent than those of Salmonella. They do not form black center.

Xylose lysine deoxycholate agar (XLD)

XLD - best selective media for Shigella - less inhibitory to S. dysenteriae and S. flexneri than DCA. Colonies are red and unlike those of most Salmonella without black centers.

 

Peptone water and Nutrient Broth

Good growth with uniform turbidity on incubation over night at 37°C. In some cases, especially fimbriated form a surface pellicle on longer incubation.

Selenite F-broth

Selenite F-broth enrich S. sonnei and S. flexneri but inhibitory to other Shigella.

[ XLD- Xylose lysine deoxycholate agar 

•        yeast extract

•        sodium chloride (NaCl)

•        xylose

•        lactose

•        sucrose

•        lysine

•        sodium thiosulfate

•        ferric ammonium citrate

•        phenol red

•        sodium deoxycholate

•        agar

•        water

sodium deoxycholate as the selective agent inhibitory to gram-positive micro-organisms. 

Xylose is fermented by practically all enterics except for the Shigella 

Lysine -Salmonella would ferment the xylose and exhaust the supply of xylose;  then, lysine is attacked via the enzyme lysine decarboxylase- creates alkaline pH – red colour (which mimics the Shigella reaction).

Sodium thiosulfate and ferric ammonium citrate in the medium, helps in the visualization of the hydrogen sulfide produced, resulting in the formation of colonies with black centers- Salmonellae

Degradation of xylose, lactose and sucrose generates acid products, causing a color change in the medium from red to yellow- E. coli 

Hydrogen sulfide production under alkaline conditions causes colonies to develop black centers- Salmonellae

Lysine decarboxylation  causes reversion to an alkaline condition and the color of the medium changes back to red- Salmonellae  

Salmonella Typhi – Red Colonies, Black Centers

Shigella sonnei – Red Colonies

Shigella flexneri – Red Colonies

Escherichia coli – Large, Flat, Yellow Colonies; some strains may be inhibited]

Antigenic structure of Shigella

Shigella are differentiated by their ‘O’ antigens into serotypes.

These are classified into 4 structures or subgroups based on a combination of biochemical and serological characteristics.

Shigella dysenteriae (Sub groupA):

1)These are mannitol non-fermenting, consists of 12 serotypes.

2)Shigella dysenteriae type-1 forms exotoxin – Shiga toxin

3)3 types of toxic activity have been demonstrated in Shigella culture filtrates.( Neurotoxicity, enterotoxicity, and cytotoxicity/verotoxin)

Shigella flexneri (Subgroup B)

Named after Flexner, who first time described first of the mannitol fermenting Shigella from Phillipines (1900).

Based on type specific and group specific antigen, they have been classified into six serotypes (1-6) and several subtypes

Shigella flexneri - belongs to group B. 

S. flexneri infections can usually be treated with antibiotics, although some strains have become resistant.

Shigella boydii (Subgroup C):

Boyd first described this strain from India (1931).

S. boydii isolates -18 serotypes have been identified.

S. boydii is the most genetically divergent species of the genus Shigella

Shigella sonnei (Subgroup D):

Isolated by Danish bacteriologist Carl Olaf Sonne (1915) in Germany.

Ferment lactose and sucrose late, indole negative.

Causes mildest form of bacillary dysentery.

Shigella sonnei together with Shigella flexneri, is responsible for 90% of shigellosis cases. 

Sub group A	Subgroup B	Subgroup C	Subgroup D
Shigella dysenteriae	Shigella flexneri - Flexner from Philippines (1900).	Shigella boydii -Boyd from India (1931)	Shigella sonnei- Carl Olaf Sonne (1915) in Germany.
Mannitol non-fermenting	Mannitol fermenting	Mannitol fermenting	Ferment mannitol, also ferment lactose and sucrose late
12 serotypes	six serotypes (1-6) and several subtypes	18 serotypes have been identified.	Shigella sonnei together with Shigella flexneri- 90% of Shigellosis cases
Type-1 forms Shiga toxin- exotoxin- Neurotoxicity, enterotoxicity & cytotoxicity/verotoxin	Usually treated with antibiotics, although some strains have become resistant.	Most genetically divergent species of the genus Shigella	Mildest form of bacillary dysentery.

Resistance

1) Shigella are killed at 56°C in one hour and by 1% phenol in 30 minutes.

2) In ice they last for 1-6 months.

3) They remain viable in moist environment.

4) In faeces they die within few hours due acidity produced by growth of coliforms.

Biochemical tests of Shigella

Carbohydrate utilization:

1)Most strains utilize sugar to produce acid but not gas though some strain S. flexneri and S. boydii form gas.

2)Glucose is fermented by almost all strains.

3)Lactose is not fermented within 24 hrs.

4) S. sonnei and some strains of S. dysenteriae produce acid from lactose after prolonged incubation.

5)Mannitol fermentation - differentiates Group A strain (which do not ferment mannitol) from group B, C and D, most strains of which ferment it.

6) Sucrose is not fermented except S. sonnei and some strains of S. flexneri.

Methyl red test: +ve

VP test: -ve

Reduce nitrate to nitrite

Catalase +ve

Indole -ve

Citrate -ve

H2S -ve

Urease -ve

KCN growth -ve

Gelatin not liquified.

Decarboxylation test:

Group A, B and C fail to decarboxylate lysine and ornithine.

S. sonnei decarboxylate ornithine but not lysine

Epidemiology

• Human beings are the only natural hosts

• Transmission through

•        Contaminated water & food

•        Contaminated fingers, flies, food/faeces, fomites (door handles, water taps, lavatory seats) - Four “F” s

•        In young male homosexuals, through gay bowel syndrome

•        Low infective dose – as low as 100 Bacilli

Pathogenicity of Shigella dysenteriae

•        Shigella cause disease by invading and replicating in cells lining the colon.

•        Adhere to the cells, invade, replicate intracellularly and spread cell-to-cell.

•        They first attach to and invade the M cells located in Peyer patches

•        Shigella lyse the phagocytic vacuole and replicate in the host cell cytoplasm- cause necrosis of epithelial cells – superficial ulcers

• Cell-to-cell passage: The bacteria are propelled through the cytoplasm to adjacent cells, with the rearrangement of actin filaments in the host cells

• Shigella survive phagocytosis by inducing programmed cell death (apoptosis).

• This process releases IL-1β- attracts polymorphonuclear leukocytes into the infected tissues.
• This destabilizes the integrity of the intestinal wall and allows the bacteria to reach the deeper epithelial cells.

 

S. dysenteriae strains produce an exotoxin, Shiga similar to the verotoxin of E. coli O157:H7.

The toxin primarily

Ø  damages the intestinal epithelium;

Ø  also damage glomerular endothelial cells, resulting in renal failure (HUS)

 

The clinical features of Shigella dysenteriae type 1 infection includes:

1) toxemia, sometimes bacteremia and severe dysentery leading to marked dehydration and protein loss

2) Inflammation and ulceration of the large intestine

3) Hemorrhage, abdominal pain and high fever

4) Death from circulatory collapse or kidney failure

 

Shigella can cause

v Shigellosis

v Other complications

v  Rectal prolapse

v Toxic megacolon

v Hemolytic-uremic syndrome  (HUS)

 Shigellosis is characterized by:

1)Abdominal cramps    2)Diarrhea    3)Fever     4)Bloody stools

5)The clinical symptoms of the disease appear 1 to 3 days after the bacteria are ingested. Lasts for 5-7 days, recovery maybe in two weeks also (patient is contagious in this period).

6) The first sign of infection is profuse watery diarrhea which is mediated by an enterotoxin

7) Invasion of the colonic mucosa by the bacteria – result in  lower abdominal cramps and tenesmus (straining to defecate- feeling to defecate even if bowels are empty), with abundant pus and blood in the stool.

8)Abundant neutrophils, erythrocytes, and mucus are found in the stool.

9)Infection is generally self-limited, although antibiotic treatment is recommended to reduce the risk of secondary spread to family members and other contacts.

10)Asymptomatic colonization of the organism in the colon develops in a small number of patients – acts as a persistent reservoir for infection.

 

Complications of Shigellosis

          In some cases, Shigellae also cause inflammation of the lining of the rectum (proctitis) or rectal prolapse.

          In rare cases (more commonly in S. dysenteriae infection), “toxic megacolon” -a deadly complication - colon becomes paralyzed, preventing bowel movements - abdominal pain and swelling, fever, weakness, and disorientation.

          Untreated, the colon may rupture and cause peritonitis, a life-threatening condition requiring emergency surgery.

 

Hemolytic Uremic Syndrome (HUS)

1) Hemolytic-uremic syndrome (HUS) is a group of blood disorders primarily in children, characterized by HUS triad -acute kidney failure, destruction of red blood cells, and low platelets – due to Shiga toxin

2)HUS can occur after S. dysenteriae type 1 infection.

3)Convulsions in children

4)It is usually complicated by severe dysentery, intravascular volume depletion, and cardiovascular collapse; has a higher morbidity and mortality rate than E. coli associated HUS

 Shiga toxin enters the bloodstream and attacks endothelial cells throughout the body, damages the lining of small blood vessels and triggers tiny blood clots resulting in HUS triad

Ø  Microangiopathic Hemolytic Anemia: The destruction of red blood cells as they are forced through damaged, narrowed vessels, leading to anemia and fatigue.

Ø  Thrombocytopenia: A low platelet count, as platelets are rapidly consumed trying to repair the damaged vessel walls.

Ø  Acute Kidney Injury (AKI): Impaired kidney function (azotemia) caused by clots clogging the small blood vessels in the kidneys.

 

 Lab diagnosis

Sample

• Fresh stool sample
• Rectal swab
• Serum

Laboratory Diagnosis

Microscopy

• Gram negative, rods, along with pus cells and RBC's.
• Non-motile, non-capsulated, non-spore forming

  

Biochemical Tests

• Oxidase negative
• Catalase positive

 

Cultural Characters

MacConkey agar: Non-lactose fermenting (except S. sonnei), large, circular, convex, smooth, and translucent.

Deoxycholate citrate agar (DCA): Colorless colonies , (non-lactose fermenting) except in the case of S. sonnei  which forms pink colonies due to late lactose fermentation.

Xylose lysine deoxycholate (XLD) agar: Colonies are red without black centers.

Salmonella-Shigella (SS) agar: Colorless colonies with no blackening

Heaktoen Enteric Agar (HEA): Green to blue- green colonies.

 

              MacConkey agar                              Deoxycholate citrate agar (DCA)              

  

                   SS Agar                                                                        XLD Agar

Serological Diagnosis

• Using monovalent and polyvalent anti-sera
• For serotyping- to know the strain and not for lab diagnosis

Treatment

• Uncomplicated Shigellosis – self-limiting – rest & rehydration must- no antibiotics for adults.
• For infants & children – oral rehydration to be ensured
• Perform antibiotic sensitivity test
• Fluoroquinolones such as ciprofloxacin or cotrimoxazole
• The treatment should be continued for 5-7 days.

�    Postinfection carriage is generally less than 3-4 weeks.

�    Mild cramps and diarrhea may continue for many days to weeks after treatment of shigellosis.

PREVENTION AND CONTROL

• No vaccine currently available.
• Personal & environmental hygiene

Prevention by

1) Use of safe drinking water

2) Chlorination of water source

3) Strict handwashing

4) Refrigeration and proper preparation and cooking of food

5) Food handlers must be treated with antibiotics and should not be involved in food preparation as long as stool cultures are positive for Shigella infection.