Materials and Methods

MATERIALS AND METHODS

Microorganisms

The microorganisms RS6 and RS10 were obtained from water and soil samples found in the salt springs near Jackson, Alabama. The samples were collected from low and high salinity sites respectively. The microorganisms were isolated by Mr. Randy Sterling from a selective complex medium containing 25% NaCl. Previous growth studies demonstrated that these halobacteria grew optimally at a salt concentration between 3% to 10% NaCl (12).

The halobacteria used were originally preserved on slants covered with mineral oil or as growth on agar plates maintained at four degrees Celsius. RS6, RS10, and two other halophilic isolates were recovered on a complex medium described below. Bacillus cereus (BC), a non-halophilic amylase producer, was used as a control bacterium and was cultured in similar medium lacking the salt components.

Growth Medium

The medium used to grow these halobacteria, Halobacterium Agar (HA), came from a formula provided by a scientific supply company (Ward’s Natural Science Establishment, Inc., Rochester, New York). The medium could be altered by not adding agar, a solidifying agent, to produce a Halobacterium Broth (HB) medium.

To avoid precipitation, the HA medium was made by separately autoclaving the salts and organic components shown above. Other additives such as forms of starches and sugars were included with the organic component of the medium as needed. Upon cooling to 55 °C, the two components were combined before pouring plates. All components of the HB medium, HA medium lacking agar, could be sterilized together. The medium was always set to a pH of 7.2 using 1M sodium hydroxide before autoclaving.

Amylase Production and Starch-Iodine Assay

The above microorganisms were plated on the appropriate solid medium containing a 1% soluble starch to detect amylase production using (BC) as a control. After allowing them to grow to reasonable size colonies, the plates were then flooded with a 1:4 dilution of Lugal’s iodine or Gram’s iodine solution. Iodine forms a dark blue complex with starch. Amylase production is detected by the absence of blue color around the colonies indicating that starch has been hydrolyzed (11).

Liquid Amylase Assays

Iodine Assay. An appropriate broth medium was made up and divided into eight separate portions. Starch was added to each portion in increasing concentrations from zero up to one percent, dispersed into tubes, and sterilized. A standard inoculum of RS6 and BC was formed from a three day old RS6 culture in HB. Ten mL of culture was used to inoculate each tube. The bacteria were then cultured at 25 °C on a shaker at 103 RPM at an angle.

The assay was set up with tubes, each of which contained 1.1 mL of Phosphate Buffer Solution (PBS; 0.01 M phosphate in pH 7.2 in 0.85% NaCl) and 0.2 mL of 1 mg/mL soluble starch. Two controls were made for the assay. The first control contained just PBS and water and was used as a negative control representing total starch hydrolysis and a blank to calibrate the spectrophotometer. The second control was a positive control and contained PBS and starch and represented the beginning starch concentration. The amylase was obtained by centrifuging RS6 and BC cells grown in appropriate medium plus additives to promote a supernatant which would serve as the source of amylase. Each tube received 100 mL of the supernatant. At various times, 1 mL of 0.25 M HCl was added to stop the amylase reaction. Finally, 0.6 mL of iodine was added to all of the tubes including the controls to form the blue starch-iodine complex. The optical density (O.D.) of each tube was then read at 600 nm in a spectrophotometer using the negative control as a blank. The disappearance of starch was determined by measuring the disappearance of the starch-iodine complex with time.

2-Cholor-4-Nitrophenyl Maltotrioside Reagent Assay. 2-chloro-4-nitrophenyl maltotrioside (CNP3) was used as a substrate in this assay. A yellow color is produced as the amylase breaks down the colorless CNP3 substrate (2). To begin the assay, an appropriate standard inoculum was prepared in the same manner as described above. Again, the appropriate medium tubes with increasing starch concentrations were inoculated, and the cells were pelleted in a centrifuge to give a supernatant containing amylase. A blank was created by placing 2.5 mL of the CNP3 reagent along with 0.5 mL of the HB medium in a tube. The assay was begun by adding 100 mL of the supernatant to 2.5 mL of the reagent and measuring the O.D. at 405 nm in the spectrophotometer with time.

Amylopectin Azure Reagent Assay. This assay consisted of the suspension of an insoluble blue starch, which amylase could degrade to release blue color into the solution (10). The inoculum, cultures, and supernatant were prepared as with the two previous assays. The assay consisted of 0.8 mL of the reagent substrate, along with 0.1 mL of supernatant or sterile medium, and 0.1 mL of 5X PBS. The O.D. at 595 nm was determined in the supernatant after pelleting the insoluble blue starch using a centrifuge. The amylase activity was recorded over time.

Growth Measurements on HA Plates and in HB Broth

Growth on Plates. The plate assays consisted of HA plates containing various additives of soluble starch, insoluble starch, and sugars. A unique inoculation method was developed to quantitate growth on solid plates by using a grid to place each individual inoculation point on the plates. A standard inoculum was used. The standard inoculum for these experiments was prepared by resuspending part of a plate colony into the appropriate medium and adjusting to an O.D. of 0.4 to 0.6 at 550 nm. In some cases, the inoculum was further diluted before inoculation. A 5 or 10 mL drop of the appropriate inoculum was consistently placed on each plate to yield a standard diameter of growth. The plates were incubated at 25 °C and placed at 4 °C to stop the growth at various time points. Growth was recorded with the scale 0 to 4+ with 0 having no growth and 4+ having maximal growth. Starch containing plates were flooded with an iodine solution to quantitate the amount of amylase hydrolysis of starch as indicated in the various tables.

Growth in Glucose Broth. An HB medium in 100 mL bottles with various glucose concentrations was used for quantitative growth measurements of RS6. A standard inoculum of RS6 was used as above for each growth medium and the bottles were incubated at 25 °C on a shaker at 100 RPM. Sterile pipettes were used to remove 3 mL of the various growths at selected time points and an O.D. was determined at 550 nm in a spectrophotometer.

The cells from the glucose concentration broth experiments were centrifuged, washed once in HB medium, and resuspended into regular HB medium to form a standard inoculum as described above. A 5 to 10 mL inoculum was placed on HA plates containing 1% glucose or no glucose as described previously.

Glucose Diffusion

In an attempt to explain the different growth data on plates and in broth, an experiment was set up to demonstrate how glucose diffuses in an HA medium. This simple experiment was based on the diffusion of antigens and antibodies in an agar medium. A sterile cork borer was used to create a hole in the middle of the medium of several HA plates. A sterile swab was used to collect RS6 cells in HB. Using the swab, the cells were spread over the HA plates, and incubated at 25 °C. Next, 100 mL of sterile 20% glucose solution was aseptically placed into the hole in the medium and replenished every 12 hours or as needed. Plates were observed with time. Examined plates were placed at four degrees Celsius for documentation.

Isolation of Glucose Resistant Cells

Four different colonies of RS6 that appeared on glucose and maltose HA media were plated on an HA medium for isolation and a single colony from each isolate was used to prepare a standard inoculum as above. HA plates containing 1% glucose or no glucose were inoculated as described above. An original standard RS6 inoculum was also used to compare to the resistant colonies. Growth was determined with time as described above. The resistant colonies were also placed on HA plates containing 1% starch for the amylase test. The starch plates were flooded with iodine to test for amylase at the appropriate time. Isolated glucose-resistant and sensitive cells were also stained with crystal violet using standard techniques (3). Cell morphology and size were determined using a calibrated ocular micrometer under the oil immersion objective.